Bicyclic nucleosides and oligomers prepared therefrom

ABSTRACT

Oligomers can be prepared from bicyclic nucleosides. The nucleosides can be a compound of formula (I) 
     
       
         
         
             
             
         
       
         
         
           
             in which each of T 1  and T 2  is independently OR 1  or OR 2 ; R 1  is H or a hydroxyl protecting group, R 2  is a phosphorus moiety; and Bx is a nucleobase. The compounds, bicyclic nucleosides and oligomers are useful for the prevention, treatment or diagnosis of muscular dystrophy.

CROSS-REFERENCED TO RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. § 371 ofInternational Application No. PCT/EP2017/080769, filed Nov. 29, 2017,designating the U.S., and published in English as WO 2018/099946 A1 onJun. 7, 2018, which claims priority to EP Application No. 16201350.2,filed Nov. 30, 2016, the entire contents of which are incorporatedherein by reference.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isP5164PC00_FINAL_SEQ Protocol ST25t.TXT, the date of creation of theASCII text file is Jan. 7, 2021, and the size of the ASCII text file is25.7 KB.

The present invention relates to novel bicyclic nucleosides andoligomers prepared therefrom. In particular, the present inventionrelates to a compound of formula (I), to an oligomer comprising at leastone compound of formula (IV), to the compounds or the oligomer of theinvention for use as a medicament in the prevention, treatment ordiagnosis of a disease and to a pharmaceutical composition comprising atleast one compound or at least one oligomer of the invention. Theinvention also refers to in vitro uses of the oligomer of the inventionfor binding to a target nucleic acid sequence and a method forsolid-phase synthesis of an oligomer of the invention.

RELATED ART

Antisense therapy has matured as an important platform for thedevelopment of innovative drugs. Oligonucleotide analogs displayingstrong and sequence specific binding to single-stranded RNA ordouble-stranded DNA and exhibiting resistance to enzymatic degradationare potential candidates for therapeutic applications as inhibitors ormodulators of protein expression. Chemically modified nucleosides areincorporated into antisense compounds to enhance its properties, such asnuclease resistance, pharmacokinetics or affinity for a target RNA.

In the recent years, the field of synthetic biology has evolved with thegeneral aim to develop antisense systems with improved potency andefficacy that do not rely on the known molecular components used innature. A basic requirement to achieve this goal is the development ofartificial genetic polymers, often referred to as xeno-nucleic acids(XNA), that fulfill the function of natural DNA or RNA (Herdewijn etal., Chem. Biodiversity 2009, 6, 791). The availability of such systemsand their integration into living organisms is expected to equip themwith novel properties that are of interest in the field of biotechnologyand medicine.

From the vast repertoire of nucleic acid modifications that appearedover the last 30 years, a handful of candidates has been scrutinizedtowards their suitability as an alternative genetic material. In anattempt to introduce non-natural components into the natural geneticmachinery by minimal chemical alteration, it has been shown thatthymidine in the genome of E. coli strains can be replaced by5-chlorouridine in an evolutionary process to a minimum residualthymidine content (Marhère et al., Angewandte Chemie, Int. Edition 2011,50, 7109). In a different approach, it has been reported that unnaturalnucleic acids, such as 1,5-anhydrohexitol nucleic acid (HNA; Hendrix etal., Chem. Eur. J. 1997, 3, 110) and cyclohexene nucleic acid (CeNA;Nauwelaerts et al., Nucleic Acids Res. 2005, 33, 2452),fluoroarabinooligonucleotides (FANA; Wilds et al., J. Nucleic Acids Res.2000, 28, 3625), arabinonucleic acids (ANA; Damha et al., J. Am. Chem.Soc. 1998, 120, 12976), threose nucleic acid (TNA; Schoning et al.,Science 2000, 290, 1347) and locked nucleic acid (LNA; Koshkin et al.,J. Am. Chem. Soc. 1998, 120, 13252; Obika et al., Tetrahedron Lett.1998, 39, 5401) can be transcribed from and reverse transcribed into DNAby DNA-polymerases.

In an alternative approach, backbone structure of RNA and DNA has beenaltered by changing the point of attachment of the internucleosidicphosphate unit to the sugar from the 3′ to the 2′ oxygen (2′,5′-DNA or2′,5′-RNA). While 2′,5′-RNA is a naturally occurring biopolymer, firstfound in bacteria in the form of 2′,5′ polyadenylates, 2′,5′-DNA isartificial (Trujillo et al., Eur. J. Biochem. 1987, 169, 167). Thepairing and replication properties of both polymers have beeninvestigated in the past. It was found that 2′,5′-RNA binds tocomplementary RNA but not to DNA. Duplexes with RNA are slightly lessstable as compared to pure RNA duplexes and duplexes in the pure2′,5′-RNA series exist but are even less stable (Wasner et al., J.Biochemistry 1998, 37, 7478). In addition, it was shown in primertemplate extension experiments that stretches of up to four 2′,5′-linkednucleotides on a template can be reverse transcribed into DNA withpolymerases or reverse transcriptases even though there is nosignificant affinity of 2′,5′-DNA to natural DNA (Sinha et al., J. Am.Chem. Soc. 2004, 126, 40).

In order to stabilize complex formation with complementary naturalnucleic acids entropically, a bicyclic DNA analog that differs fromnatural DNA by an additional ethylene bridge located between the centersC(3′) and C(5′) has been designed. The points of attachment of thelinking phosphodiester units are the same as in the naturally occurringnucleic acid, i.e. the 3′ and 5′ terminus. This change in the carbonskeleton results in a locked sugar conformation which causes thebicyclo-deoxynucleosides to exhibit a higher degree of preorganisationof the single strands for duplex formation. Decamers ofbicyclo-deoxyadenosine and bicyclo-thymidine bind to their natural RNAand DNA complements as well as with each other, forming double andtriple helical structures. Compared with natural DNA, duplex formationis associated with reduced pairing enthalpy and entropy terms, havingcompensatory effects on the free energy of duplex formation (Bolli etal., Nucleic Acids Res. 1996, 24, 4660).

Nevertheless, after more than three decades of research in the antisensefield, clinical applications are still limited by the low biostability,the poor pharmacokinetic properties and the off-target toxicity of thisclass of compounds.

Thus, there remains a need for antisense compounds that are resistant toenzymatic degradation in vivo and display strong and sequence specificbinding to nucleic acids.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a compound of formula (I):

wherein one of T₁ and T₂ is OR₁ or OR₂;and the other of T₁ and T₂ is OR₁ or OR₂; whereinR₁ is H or a hydroxyl protecting group, andR₂ is a phosphorus moiety; and whereinBx is a nucleobase.

In a second aspect, the invention provides an oligomer comprising atleast one compound of formula (IV)

wherein independently for each of said at least one compound of formula(IV)one of T₃ or T₄ is a nucleosidic linkage group;the other of T₃ and T₄ is OR₁, OR₂, a 5′ terminal group, a 7′ terminalgroup or a nucleosidic linkage group, wherein R₁ is H or a hydroxylprotecting group, and R₂ is a phosphorus moiety; and Bx is a nucleobase.

In a third aspect, the present invention provides for the inventivecompound of formula (I), (II) or (III) or the oligomer of the inventionfor use as a medicament in the prevention or treatment of a disease.

In a further aspect, the present invention provides for the oligomer ofthe invention, preferably the inventive oligomer of the formula (V), foruse as a medicament in the prevention, treatment or diagnosis of adisease, wherein said disease is a muscular dystrophy, and whereinpreferably said disease is Duchenne muscular dystrophy.

In a further aspect, the invention provides a pharmaceutical compositioncomprising at least one compound selected from formula (I), (II) or(III) or at least one oligomer of the invention.

In a further aspect, the oligomer of the invention is used in vitro forbinding to a target nucleic acid sequence.

In a further aspect, the invention provides a method for solid-phasesynthesis of an oligomer of the invention.

The invention described herein provides for new compounds, wherein theposition of the group used for linkage to other entities, such as thenucleosidic linkage group, is shifted as compared to compounds of theprior art. Thus, the compounds of the invention can be or are linked viathe 5′ terminus and 7′ terminus of its bicyclic sugar to other entitiessuch as nucleosides or nucleotides (FIG. 1C and FIG. 1D). In contrastand in the prior art, nucleosides or nucleotides are linked via the 3′and 5′ termini, be it for known nucleosides or nucleotides comprising abicyclic sugar (FIG. 1B) or as in naturally occurring DNA or RNA (FIG.1A).

As a consequence of that shifted linkage, the backbone geometry of thecompounds of the invention within an oligomer is changed. This changedbackbone geometry in turn induces a nucleobase stacking of the compoundsof the invention that is different from that of naturally occurringnucleic acid helixes. Consequently, oligomers of the invention adoptshelix conformations that are distinctly different from that of naturalDNA. Furthermore, based on these findings, oligomers of the inventionforming duplex structures are expected to have a geometry deviating fromconventional canonical duplexes.

Despite of the changes in backbone geometry and nucleobase stacking, wehave now surprisingly found that oligomers of the invention cross-pairwith natural DNA and RNA, and self-duplexes of oligomers of theinvention exhibit thermal stabilities that are in the same range as thatof natural DNA duplexes. Consequently, the oligomers of the inventionhave the structural and base-pairing properties necessary to become anovel xeno-nucleic acid that may fulfill the function of natural DNA.Moreover, the oligomers of the invention show comparable base pairingselectivity and have even a better mismatch discrimination abilitycompared to its natural DNA counterpart. Increasing the mismatchdiscrimination typically reduces the potential off-target effects andtherefore represents an appealing property for an antisense candidate.

DESCRIPTION OF FIGURES

FIG. 1: A) α-monocyclic DNA; B) bicyclic (bc-)DNA; C) 7′,5′-β-bc-DNA,i.e. the inventive compound of formula (III); D) 7′,5′-α-bc-DNA, i.e.the inventive compound of formula (II).

FIG. 2: Comparison between 7′,5′-β-bc-DNA, i.e. the inventive compoundof formula (III) depicted on the left side, and 7′,5′-α-bc-DNA, i.e. theinventive compound of formula (II) depicted on the right side.

FIG. 3: X-ray structure of a) 5′-O-p-nitrobenzoyl-7′,5′-α-bc-T, b)5′-O-acetyl-7′,5′-α-bc-GAc. Non-polar hydrogen atoms are omitted forclarity.

FIG. 4: Insertion of 7′,5′-α-bc-DNA with polarity reversal inside β-DNA.

FIG. 5: UV-melting curves (260 nm) of oligonucleotide ON21 (SEQ ID NO:21) with fully modified parallel (oligonucleotide ON22; SEQ ID NO: 22)and antiparallel (oligonucleotide ON23; SEQ ID NO: 23) complement,parallel DNA and parallel RNA, in comparison with the correspondingnatural DNA duplex. Total strand concentration: 2 μM in 10 mM NaH₂PO₄,150 mM NaCl, pH 7.0.

FIG. 6: CD-spectra of duplexes a) DNA.RNA, b) ON21.RNA and c) ON21.DNAd) ON21.ON22 at 20° C. Experimental conditions: Total strand conc. 2 μMin 10 mM NaH2PO4, 150 mM NaCl, pH 7.0.

FIG. 7: Cropped picture of a gel. a) DNA control experiment. DNAdigestion reaction after b) 1 hour c) 2 hours d) 4 hours e) 24 hours; f)control experiment with oligonucleotide ON21; ON21 digestion reactionafter g) 1 hour h) 2 hours i) 4 hours j) 24 hours.

FIG. 8: Results of C3 complement activation; PO denotes phosphatenucleosidic linkages, PS denotes phosphorothioate nucleosidic linkages,bc-DNA denotes the 7′,5′-α-bc-DNA scaffold, tc-DNA denotes the tricycloscaffold. Each measurement has been repeated at least 3 times. Thesequence is similar for the 5 ONs.

FIG. 9: Comparison of the mRNA expression level after incubation withPS-α-bc-DNA or PS-tc-DNA. Sequence is similar for the two ONs.Abbreviations as mentioned in FIG. 8.

FIG. 10: X-ray structure of 7′-O-p-nitrobenzoyl-7′,5′-β-bc-T. Hydrogenatoms are omitted for clarity.

FIG. 11: UV-melting curves (260 nm) of the homo 7′,5′-β-bc-DNA duplex incomparison with the corresponding natural DNA duplex. Total strandconcentration: 2 μM in 10 mM NaH₂PO₄, 150 mM NaCl, pH 7.0.

FIG. 12: CD-spectra of the three duplexes a) ON13.ON14, b) ON13.DNA andc) DNA.DNA at temperatures between 10 and 80° C. Experimentalconditions: Total strand concentration 2 μM in 10 mM NaH₂PO₄, 1 M NaCl,pH 7.0, 10° C.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs.

The terms “protecting group for an amino”, “protecting group for anamino group”, or “amino protecting group” as interchangeably usedherein, are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, Greene's ProtectiveGroups in Organic Synthesis, P. G. M. Wuts, 5th edition, John Wiley &Sons, 2014, and in Current Protocols in Nucleic Acid Chemistry, editedby S. L. Beaucage et al. 06/2012, and hereby in particular in Chapter 2.Suitable “amino protecting groups” for the present invention include andare typically and preferably independently at each occurrence selectedfrom methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate(Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate(TCBOC), benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz) and2,4,6-trimethylbenzyl carbamate; as well as formamide, acetamide,benzamide.

The terms “protecting group for a hydroxyl”, “protecting group for ahydroxyl group”, or “hydroxyl protecting group” as interchangeably usedherein, are well known in the art and includes those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999; Greene's ProtectiveGroups in Organic Synthesis, P. G. M. Wuts, 5^(th) edition, John Wiley &Sons, 2014, and in Current Protocols in Nucleic Acid Chemistry, editedby S. L. Beaucage et al. 06/2012, and hereby in particular in Chapter 2.In a certain embodiment, the “hydroxyl protecting groups” of the presentinvention include and, typically and preferably are independently ateach occurrence selected from, acetyl, benzoyl, benzyl,β-methoxyethoxymethyl ether (MEM), dimethoxytrityl,[bis-(4-methoxyphenyl)phenylmethyl] (DMTr), methoxymethyl ether (MOM),methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzylether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl(THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ether,such as t-Butyldiphenylsilyl ether (TBDPS), trimethylsilyl (TMS),tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), andtriisopropylsilyl (TIPS) ethers; methyl ethers, ethoxyethyl ethers (EE).

In a preferred embodiment, the “hydroxyl protecting groups” of thepresent invention include and, typically and preferably areindependently at each occurrence selected from, acetyl, t-butyl,t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl,diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl),4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl,t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS),triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl,trichloroacetyl, trifiuoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate,mesylate, tosylate, triflate, 4-monomethoxytrityl (MMTr),4,4′dimethoxytrityl, (DMTr) and 4,4′,4″-trimethoxytrityl (TMTr),2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE),2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl(NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl,2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl,2-(2-nitrophenyl)ethyl, butylthiocarbonyl,4,4′,4″-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl,2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl(Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-y1 (MOX).

In some embodiments, the hydroxyl protecting group is independently ateach occurrence selected from acetyl, benzyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, trityl, 4-monomethoxytrityl, 4,4′-dimethoxytrityl(DMTr), 4,4′,4″-trimethoxytrityl (TMTr), 9-phenylxanthin-9-yl (Pixyl)and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In preferred embodiments, thehydroxyl protecting group is independently at each occurrence selectedfrom triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4′-dimethoxytrityl(DMTr), 4,4′,4″-trimethoxytrityl (TMTr), 9-phenylxanthin-9-yl (Pixyl)and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In further preferredembodiments, the hydroxyl protecting group is independently at eachoccurrence selected from trityl, 4-monomethoxytrityl and4,4′-dimethoxytrityl group. In a very preferred embodiment, saidhydroxyl protecting group is independently at each occurrence selectedfrom triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4′-dimethoxytrityl(DMTr), 4,4′,4″-trimethoxytrityl (TMTr), 9-phenylxanthin-9-yl (Pixyl)and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In a more preferredembodiment, the hydroxyl protecting groups of the present invention isacetyl, dimethoxytrityl (DMTr), tert-butyldimethylsilyl (TBDMS),tri-iso-propylsilyloxymethyl (TOM), or t-butyldiphenylsilyl ether(TBDPS). In an again very preferred embodiment, said hydroxyl protectinggroup is independently at each occurrence selected from4,4′-dimethoxytrityl (DMTr) or 4-monomethoxytrityl. In an again furthervery preferred embodiment, said hydroxyl protecting group is4,4′-dimethoxytrityl (DMTr).

The term “phosphorus moiety”, as used herein, refers to a moietycomprising a phosphorus atom in the P^(III) or P^(V) valence state andwhich is represented by formula (VII)

wherein

W represents O, S or Se or W represents an electron pair;

R₃ and R₄ are independently of each other H, halogen, OH, OR₅, NR₆R₇,SH, SR₈, C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,C₁-C₆aminoalkyl; wherein R₅ is C₁-C₉alkyl, C₁-C₆alkoxy, eachindependently of each other optionally substituted with cyano, nitro,halogen, —NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl;aryl, C₁-C₆alkylenearyl, C₁-C₆alkylenediaryl, each independently of eachother optionally substituted with cyano, nitro, halogen, C₁-C₄alkoxy,C₁-C₄haloalkyl, C₁-C₄haloalkoxy, NHC(O)C₁-C₃alkyl, NHC(O)C₁-C₃haloalkyl,C₁-C₃alkylsulfonyl; acetyl; a hydroxyl protecting group; wherein R₆ andR₇ are independently of each other hydrogen, C₁-C₉alkyl optionallysubstituted with cyano, nitro, halogen, C₂-C₆alkenyl, C₃-C₆cycloalkyl,C₁-C₃alkoxy; aryl optionally substituted with cyano, nitro, halogen,C₁-C₃alkyl, C₁-C₃alkoxy; an amino protecting group; or together with thenitrogen atom to which they are attached form a heterocyclic ring,wherein preferably said heterocyclic ring is selected from pyrollidinyl,piperidinyl, morpholinyl, piperazinyl and homopiperazine, wherein saidheterocyclic ring is optionally substituted with C₁-C₃ alkyl; andwherein R₈ is a thiol protecting group; and wherein the wavy lineindicates the attachment to the oxygen of said OR₂ group. When Wrepresents O, S or Se then said P atom within said phosphorus moiety isin its P^(V) valence state. When W represents an electron pair then saidP atom within said phosphorus moiety is in its P^(III) valence. Themoiety of formula (VII) includes any possible stereoisomer. Furtherincluded in said moieties represented by formula (VII) are saltsthereof, wherein typically and preferably said salts are formed upontreatment with inorganic bases or amines, and are typically andpreferably salts derived from reaction with the OH or SH groups being(independently of each other) said R₃ and R₄. Preferred inorganic basesor amines leading to said salt formation with the OH or SH groups arewell known in the art and are typically and preferably trimethylamine,diethylamine, methylamine or ammonium hydroxide. These phosphorusmoieties included in the present invention are, if appropriate, alsoabbreviated as “O⁻HB⁺”, wherein said HB⁻ refers to the counter cationformed.

In a preferred embodiment, in the “phosphorus moiety”, R₃ and R₄ areindependently of each other H, OH, OR₅, NR₆R₇, C₁-C₆alkyl, C₁-C₆alkyl,C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy, C₁-C₆aminoalkyl; whereinR₅ is C₁-C₉alkyl optionally substituted with cyano, nitro, halogen;aryl, C₁-C₆alkylenearyl, each independently of each other optionallysubstituted with cyano, nitro, halogen; acetyl; a hydroxyl protectinggroup; wherein R₆ and R₇ are independently of each other hydrogen,C₁-C₉alkyl optionally substituted with cyano, nitro, halogen; aryloptionally substituted with cyano, nitro, halogen, C₁-C₃ alkyl,C₁-C₃alkoxy; an amino protecting group; and wherein R₈ is a thiolprotecting group; and wherein the wavy line indicates the attachment tothe oxygen of said OR₂ group.

The term “phosphorus moiety”, as used herein, includes and, typicallyand preferably is independently at each occurrence selected from amoiety derived from phosphonates, phosphite triester, monophosphate,diphosphate, triphosphate, phosphate triester, phosphate diester,thiophosphate ester, di-thiophosphate ester or phosphoramidites.

Thus, in a preferred embodiment, said OR₂ is independently at eachoccurrence selected from phosphonates, phosphite triester,monophosphate, diphosphate, triphosphate, phosphate triester, phosphatediester, thiophosphate ester, di-thiophosphate ester orphosphoramidites, and wherein preferably said OR₂ is a phosphoramiditeor a phosphate triester, more preferably a phosphoramidite.

In a preferred embodiment, the phosphorus moiety is derived from aphosphonate represented by formula (VII), wherein W is O, R₃ is selectedfrom C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,C₁-C₆aminoalkyl, and R₄ is OH or O⁻HB⁺; and wherein the wavy lineindicates the attachment to the oxygen of said OR₂ group. In anotherembodiment, the phosphorus moiety of formula (VII) is an H-phosphonate,wherein W is O, R₃ is hydrogen and R₄ is OH or O⁻HB⁺; and whereinpreferably said O⁻HB⁺ is HNEt₃ ⁺. In a further embodiment, thephosphorus moiety of formula (VII) is an alkyl-phosphonate, wherein W isO, R₃ is alkyl, and R₄ is OH or O⁻HB⁺; and wherein preferably said O⁻HB⁺is HNEt₃ ⁺. More preferably, the phosphorus moiety of formula (VII) ismethyl-phosphonate, wherein W is O, R₃ is hydrogen and R₄ is OH orO⁻HB⁺; and wherein preferably said O⁻HB⁺ is HNEt₃ ⁺). In anotherembodiment, the phosphorus moiety of formula (VII) is aphosphonocarboxylate, wherein R₃ or R₄ are independently of each other acarboxylic acid. Preferably, said phosphonocarboxylate isphosphonoacetic acid or phosphonoformic acid. In a further embodiment,the phosphorus moiety of formula (VII) is a 2-aminoethyl-phosphonate.

In a preferred embodiment, R₃ and R₄ of the phosphorus moiety of formula(VII) are independently of each other H, OH, halogen, OR₅, NR₆R₇, SH,SR₈, C₁-C₄alkyl, preferably C₁-C₂alkyl, C₁-C₄haloalkyl, preferablyC₁-C₂haloalkyl, C₁-C₄alkoxy, preferably C₁-C₂alkoxy, C₁-C₄haloalkoxy,preferably C₁-C₂haloalkoxy, C₁-C₄aminoalkyl, preferably C₁-C₂aminoalkyl;and wherein R₅ is C₁-C₆alkyl, preferably C₁-C₃alkyl, each independentlyof each other optionally substituted with cyano, nitro, halogen,NHC(O)C₁-C₃alkyl, NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; aryl,C₁-C₃alkylenearyl, C₁-C₃alkylenediaryl, each independently of each otheroptionally substituted with cyano, nitro, halogen, C₁-C₄alkoxy,C₁-C₄haloalkyl, C₁-C₄haloalkoxy, NHC(O)C₁-C₃alkyl, NHC(O)C₁-C₃haloalkyl,C₁-C₃alkylsulfonyl; acetyl; a hydroxyl protecting group; and wherein R₆and R₇ are independently of each other hydrogen, C₁-C₆alkyl, preferablyC₁-C₄alkyl, each independently of each other optionally substituted withcyano, nitro, halogen, C₂-C₄alkenyl, C₃-C₆cycloalkyl, C₁-C₃alkoxy; aryloptionally substituted with cyano, nitro, halogen, C₁-C₃ alkyl,C₁-C₃alkoxy; an amino protecting group; or together with the nitrogenatom to which they are attached form a heterocyclic ring, whereinpreferably said heterocyclic ring is selected from pyrollidinyl,piperidinyl, morpholinyl, piperazinyl and homopiperazine, wherein saidheterocyclic ring is optionally substituted with C₁-C₃ alkyl; andwherein R₈ is a thiol protecting group; and wherein the wavy lineindicates the attachment to the oxygen of said OR₂ group.

In another preferred embodiment, R₃ or R₄ of the phosphorus moiety offormula (VII) is independently at each occurrence and of each otherhalogen, preferably chlorine, or OR₅, wherein R₅ is a hydroxylprotecting group. Additional phosphorus moieties used in the inventionare disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer,Tetrahedron, 1992, 48, 2223-2311).

The term “phosphorus moiety”, as used herein, preferably refers to agroup R₂ comprising a phosphorus atom in the P^(III) or P^(V) valencestate and which is represented independently at each occurrence eitherby formula (VIII), formula (IX) or formula (X),

wherein Y is O, S or Se, and wherein Y preferably is O or S, morepreferably Y is O; and wherein R₅ and R_(5′) are independently at eachoccurrence and of each other hydrogen, C₁-C₉alkyl, C₁-C₆alkoxy, eachindependently of each other optionally substituted with cyano, nitro,halogen, —NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl;aryl, C₁-C₆alkylenearyl, C₁-C₆alkylenediaryl each independently of eachother optionally substituted with cyano, nitro, halogen, C₁-C₄alkoxy,C₁-C₄haloalkyl, C₁-C₄haloalkoxy, —NHC(O)C₁-C₃alkyl,NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; a hydroxyl protecting group;wherein R₆ and R₇ are independently of each other hydrogen, C₁-C₉alkyloptionally substituted with cyano, nitro, halogen, C₂-C₆alkenyl,C₃-C₆cycloalkyl, C₁-C₃alkoxy; aryl, preferably phenyl, optionallysubstituted with cyano, nitro, halogen, C₁-C₃ alkyl, C₁-C₃alkoxy; anamino protecting group; or together with the nitrogen atom to which theyare attached form a heterocyclic ring, wherein preferably saidheterocyclic ring is selected from pyrollidinyl, piperidinyl,morpholinyl, piperazinyl and homopiperazine, wherein said heterocyclicring is optionally substituted with C₁-C₃ alkyl; and wherein R₈ is athiol protecting group; and wherein the wavy line indicates theattachment to the oxygen of said OR₂ group.

In a preferred embodiment, said phosphorus moiety R₂ is represented byformula (VIII)

wherein Y is O, S or Se, wherein Y preferably is O or S, most preferablyY is O; and wherein R₅ and R_(5′) are independently at each occurrenceand of each other hydrogen, C₁-C₉alkyl, C₁-C₆alkoxy, each independentlyof each other optionally substituted with cyano, nitro, halogen,—NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; aryl,C₁-C₆alkylenearyl, C₁-C₆alkylenediaryl each independently of each otheroptionally substituted with cyano, nitro, halogen, C₁-C₄alkoxy,C₁-C₄haloalkyl, C₁-C₄haloalkoxy, —NHC(O)C₁-C₃alkyl,NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; a hydroxyl protecting group;P(O)(OR₉)(OR_(9′)), P(O)OP(O)(OR₉)(OR_(9′)); wherein R₉ and R_(9′) areindependently at each occurrence and of each other hydrogen, C₁-C₉alkyloptionally substituted with cyano, nitro, halogen, —NHC(O)C₁-C₃alkyl,—NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; aryl, C₁-C₆alkylenearyl,C₁-C₆alkylenediaryl each independently of each other optionallysubstituted with cyano, nitro, halogen, C₁-C₄alkoxy, C₁-C₄haloalkyl,C₁-C₄haloalkoxy, —NHC(O)C₁-C₃alkyl, NHC(O)C₁-C₃haloalkyl,C₁-C₃alkylsulfonyl; a hydroxyl protecting group; and wherein the wavyline indicates the attachment to the oxygen of said OR₂ group.

In a preferred embodiment, R₅ and R_(5′) of formula (VIII) areindependently at each occurrence and of each other hydrogen, C₁-C₆alkyl,preferably C₁-C₃alkyl, C₁-C₄alkoxy, preferably C₁-C₂alkoxy, eachindependently of each other optionally substituted with cyano, nitro,halogen, —NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl;aryl, preferably phenyl, C₁-C₄alkylenearyl, C₁-C₄alkylenediaryl eachindependently of each other optionally substituted with cyano, nitro,halogen, C₁-C₄alkoxy, C₁-C₄haloalkyl, C₁-C₄haloalkoxy,—NHC(O)C₁-C₃alkyl, NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; a hydroxylprotecting group.

In a preferred embodiment, R₅ and R_(5′) of formula (VIII) areindependently of each other C₁-C₄alkyl or aryl, preferably phenyl. Inanother preferred embodiment, R₅ and R_(5′) of formula (VIII) areindependently of each other methyl or ethyl. In a further preferredembodiment, R₅ and R_(5′) of formula (VIII) are independently of eachother phenyl or benzyl. In another preferred embodiment, R₅ and R_(5′)are independently at each occurrence and of each other hydrogen or ahydroxyl protecting group, preferably a hydroxyl protecting group. In apreferred embodiment, in formula (VIII), R₅ and R_(5′) are independentlyat each occurrence and of each other hydrogen, C₁-C₉alkyl, C₁-C₆alkoxy,each independently of each other optionally substituted with cyano,nitro, halogen; aryl, C₁-C₆alkylenearyl, each independently of eachother optionally substituted with cyano, nitro, halogen; or a hydroxylprotecting group. Typically and preferably, said phosphorus moiety R₂represented by formula (VIII) is herein referred as “phosphate moiety”.

In a preferred embodiment, said phosphorus moiety R₂ is represented byformula (IX)

wherein

wherein

Y is O, S or Se, and wherein Y preferably is O or S, most preferably Yis O; and wherein

R₅ is independently at each occurrence hydrogen, C₁-C₉alkyl,C₁-C₆alkoxy, each independently of each other optionally substitutedwith cyano, nitro, halogen, —NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl,C₁-C₃alkylsulfonyl; aryl, C₁-C₆alkylenearyl, C₁-C₆alkylenediaryl eachindependently of each other optionally substituted with cyano, nitro,halogen, C₁-C₄alkoxy, C₁-C₄haloalkyl, C₁-C₄haloalkoxy,—NHC(O)C₁-C₃alkyl, NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; a hydroxylprotecting group; wherein

R₆ and R₇ are independently of each other hydrogen, C₁-C₉alkyloptionally substituted with cyano, nitro, halogen, C₂-C₆alkenyl,C₃-C₆cycloalkyl, C₁-C₃alkoxy; aryl, preferably phenyl, optionallysubstituted with cyano, nitro, halogen, C₁-C₃ alkyl, C₁-C₃alkoxy; anamino protecting group; or together with the nitrogen atom to which theyare attached form a heterocyclic ring, wherein preferably saidheterocyclic ring is selected from pyrollidinyl, piperidinyl,morpholinyl, piperazinyl and homopiperazine, wherein said heterocyclicring is optionally substituted with C₁-C₃ alkyl; and wherein the wavyline indicates the attachment to the oxygen of said OR₂ group. Typicallyand preferably, said phosphorus moiety R₂ represented by formula (IX) isreferred herein as “phosphoramidate moiety” or, interchangeably used,“phosphoroamidate moiety”.

In a preferred embodiment, said phosphorus moiety R₂ is represented byformula (X)

wherein

R₅ is hydrogen, C₁-C₉alkyl, C₁-C₆alkoxy, each independently of eachother optionally substituted with cyano, nitro, halogen,—NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; aryl,C₁-C₆alkylenearyl, C₁-C₆alkylenediaryl independently of each otheroptionally substituted with cyano, nitro, halogen, C₁-C₄alkoxy,C₁-C₄haloalkyl, C₁-C₄haloalkoxy, —NHC(O)C₁-C₃alkyl,NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl, a hydroxyl protecting group;and wherein

R₆ and R₇ are independently of each other hydrogen, C₁-C₉alkyloptionally substituted with cyano, nitro, halogen, C₂-C₆alkenyl,C₃-C₆cycloalkyl, C₁-C₃alkoxy, aryl, preferably phenyl, optionallysubstituted with cyano, nitro, halogen, C₁-C₃ alkyl, C₁-C₃alkoxy; ortogether with the nitrogen atom to which they are attached form aheterocyclic ring, wherein preferably said heterocyclic ring is selectedfrom pyrollidinyl, piperidinyl, morpholinyl, piperazinyl andhomopiperazine, wherein said heterocyclic ring is optionally substitutedwith C₁-C₃ alkyl, and wherein the wavy line indicates the attachment tothe oxygen of said OR₂ group. Typically and preferably, said phosphorusmoiety R₂ represented by formula (X) is referred herein as“phosphoramidite moiety” or, interchangeably used, “phosphoroamiditemoiety”.

In a preferred embodiment, in formula (IX) said Y is O; said R₅ isindependently at each occurrence hydrogen, C₁-C₉alkyl, C₁-C₆alkoxy, eachindependently of each other optionally substituted with cyano, nitro,halogen; aryl, C₁-C₆alkylenearyl, each independently of each otheroptionally substituted with cyano, nitro, halogen; a hydroxyl protectinggroup; wherein R₆ and R₇ are independently of each other hydrogen,C₁-C₉alkyl optionally substituted with cyano, nitro, halogen,C₂-C₆alkenyl; aryl optionally substituted with cyano, nitro, halogen,C₁-C₃ alkyl, C₁-C₃alkoxy; an amino protecting group; and wherein thewavy line indicates the attachment to the oxygen of said OR₂ group.

In a preferred embodiment, in formula (X) said R₅ is independently ateach occurrence hydrogen, C₁-C₉alkyl, C₁-C₆alkoxy, each independently ofeach other optionally substituted with cyano, nitro, halogen; aryl,C₁-C₆alkylenearyl, each independently of each other optionallysubstituted with cyano, nitro, halogen; a hydroxyl protecting group;wherein R₆ and R₇ are independently of each other hydrogen, C₁-C₉alkyloptionally substituted with cyano, nitro, halogen, C₂-C₆alkenyl; aryloptionally substituted with cyano, nitro, halogen, C₁-C₃ alkyl,C₁-C₃alkoxy; an amino protecting group; and wherein the wavy lineindicates the attachment to the oxygen of said OR₂ group.

In a very preferred embodiment, said phosphorus moiety R₂ isindependently at each occurrence selected from a phosphate moiety,phosphoramidate moiety and phosphoramidite moiety.

In a further preferred embodiment, said R₅ is independently at eachoccurrence hydrogen, C₁-C₆alkyl, preferably C₁-C₄alkyl, C₁-C₄alkoxy,each independently of each other optionally substituted with cyano,nitro, halogen, —NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl,C₁-C₃alkylsulfonyl; aryl, C₁-C₄alkylenearyl, C₁-C₄alkylenediaryl eachindependently of each other optionally substituted with cyano, nitro,halogen, C₁-C₄alkoxy, C₁-C₄haloalkyl, C₁-C₄haloalkoxy,—NHC(O)C₁-C₃alkyl, NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; a hydroxylprotecting group; wherein R₆ and R₇ are independently of each otherhydrogen, C₁-C₆alkyl optionally substituted with cyano, nitro, halogen,C₂-C₄alkenyl, C₃-C₆cycloalkyl, C₁-C₃alkoxy; aryl optionally substitutedwith cyano, nitro, halogen, C₁-C₃ alkyl, C₁-C₃alkoxy; an aminoprotecting group; or together with the nitrogen atom to which they areattached form a heterocyclic ring, wherein preferably said heterocyclicring is selected from pyrollidinyl, piperidinyl, morpholinyl,piperazinyl and homopiperazine, wherein said heterocyclic ring isoptionally substituted with C₁-C₃ alkyl; and wherein the wavy lineindicates the attachment to the oxygen of said OR₂ group.

In a further preferred embodiment, said R₅ is C₁-C₃alkyl optionallysubstituted with cyano, chlorine, fluorine or bromine; aryl,C₁-C₃alkylenearyl, C₁-C₃alkylenediaryl, each independently of each otheroptionally substituted with cyano, nitro, chlorine, fluorine, bromine,C₁-C₂alkoxy, C₁haloalkyl. In a more preferred embodiment, said R₅ is aC₁-C₃alkyl optionally and preferably substituted with cyano, chlorine,fluorine or bromine; preferably substituted with cyano. In again a morepreferred embodiment, said R₅ is a cyano substituted C₂alkyl, preferablysaid R₅ is —CH₂CH₂—CN.

In a further preferred embodiment, said R₅ is C₁-C₄alkyl, preferablymethyl or ethyl; aryl, preferably phenyl or benzyl; chloride or ahydroxyl protecting group. In a further preferred embodiment, said R₅ ismethyl or a hydroxyl protecting group.

In a further preferred embodiment, said R₅ is C₁-C₆alkoxy optionallysubstituted with cyano, chlorine, fluorine or bromine.

In a further preferred embodiment, said R₆ and R₇ are independently ofeach other H or C₁-C₃alkyl; or together with the nitrogen atom to whichthey are attached form a heterocyclic ring, wherein said heterocyclicring is selected from pyrollidinyl, piperidinyl, morpholinyl,piperazinyl wherein said heterocyclic ring is optionally substitutedwith methyl. In a further preferred embodiment, said R₆ and R₇ areindependently of each other C₁-C₃alkyl, alkoxy or aryl, wherein the arylis preferably phenyl or benzyl, optionally substituted with cyano,nitro, chlorine, fluorine, bromine. In a further preferred embodiment,said R₆ is hydrogen, and R₇ is (i) C₁-C₉alkyl or (ii) aryl, (i) or (ii)optionally substituted with cyano, nitro, halogen, aryl, whereinpreferably R₇ is C₁-C₃alkyl, phenyl or benzyl.

In a further preferred embodiment, said R₆ and R₇ are independently ofeach other selected from methyl, ethyl, isopropyl or isobutyl. In a morepreferred embodiment, said R₆ and R₇ are independently of each otherisopropyl.

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein said R₅ is (i) C₁-C₉alkyl; (ii)aryl, preferably phenyl; or (iii) said (i) or said (ii) optionallysubstituted with cyano, nitro, halogen, aryl; and wherein said R₆ and R₇are independently of each other C₁-C₉alkyl, preferably isopropyl.

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein R₅ is C₁-C₉alkyl optionallysubstituted with cyano, nitro, halogen, —NHC(O)C₁-C₃alkyl,—NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; aryl, C₁-C₆alkylenearyl,C₁-C₆alkylenediaryl independently of each other optionally substitutedwith cyano, nitro, halogen, C₁-C₄alkoxy, C₁-C₄haloalkyl,C₁-C₄haloalkoxy, —NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl,C₁-C₃alkylsulfonyl; and R₆ and R₇ are independently of each otherC₁-C₉alkyl optionally substituted with cyano, nitro, halogen,C₂-C₆alkenyl, C₃-C₆cycloalkyl, C₁-C₃alkoxy, phenyl optionallysubstituted with cyano, nitro, halogen, C₁-C₃ alkyl, C₁-C₃alkoxy; ortogether with the nitrogen atom to which they are attached form aheterocyclic ring, wherein preferably said heterocyclic ring is selectedfrom pyrollidinyl, piperidinyl, morpholinyl, piperazinyl andhomopiperazine, wherein said heterocyclic ring is optionally substitutedwith C₁-C₃ alkyl; and wherein the wavy line indicates the attachment tothe oxygen of said OR₂ group.

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein said R₅ is C₁-C₉alkyl optionallysubstituted with cyano, nitro, chlorine, fluorine, bromine,—NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl; aryl, C₁-C₆alkylenearyl,C₁-C₆alkylenediaryl independently of each other optionally substitutedwith cyano, nitro, chlorine, fluorine, bromine, C₁-C₄alkoxy,C₁-C₄haloalkyl.

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein said R₅ is C₁-C₃alkyl optionallysubstituted with cyano, chlorine, fluorine and bromine; aryl,C₁-C₃alkylenearyl, C₁-C₃alkylenediaryl, independently of each otheroptionally substituted with cyano, nitro, chlorine, fluorine, bromine,C₁-C₂alkoxy, C₁haloalkyl.

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein said R₅ is C₁-C₃alkyl, 2-cyanoethyl,2,2,2-trichloroethyl, 2,2,2-tribromoethyl, —(CH₂)—NHC(O)CF₃ whereinn=3-6; phenyl, C₁-C₃alkylenephenyl, benzhydryl, independently of eachother optionally substituted with cyano, nitro, chlorine, fluorine,bromine, C₁-C₂alkoxy, —CF₃.

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein said R₅ is methyl, ethyl,2-cyanoethyl, again preferably 2-cyanoethyl (CH₂)₂CN).

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein said R₆ and R₇ are independently ofeach other C₁-C₃alkyl or together with the nitrogen atom to which theyare attached form a heterocyclic ring, wherein said heterocyclic ring isselected from pyrollidine, piperidine, morpholine, wherein saidheterocyclic ring is optionally substituted with C₁-C₃ alkyl, andwherein again further preferably said heterocyclic ring is optionallysubstituted with methyl.

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein R₆ is equal to R₇ and R₆ and R₇ areiso-propyl or methyl.

In another very preferred embodiment, said phosphorus moiety R₂ isrepresented by formula (X), wherein said R₅ is methyl, ethyl,2-cyanoethyl, preferably 2-cyanoethyl, and wherein R₆ is equal to R₇ andR₆ and R₇ are iso-propyl or methyl.

Each alkyl moiety either alone or as part of a larger group such asalkoxy or alkylene is a straight or branched chain and is preferablyC₁-C₆alkyl, more preferably C₁-C₃alkyl. Examples include methyl, ethyl,n-propyl, prop-2-yl (iso-propyl; interchangeably abbreviated herein asiPr or Fri, in particular in the drawn chemical formula), n-butyl,but-2-yl, 2-methyl-prop-1-yl or 2-methyl-prop-2-yl. Examples of analkoxy include methoxy, ethoxy, propoxy, iso-propoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, neo-pentoxy, n-hexoxy. As describedherein, alkoxy may include further substitutents such as halogen atomsleading to haloalkoxy moieties.

Each alkylene moiety is a straight or branched chain and is, forexample, —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(CH₃)—CH₂—, or—CH(CH₂CH₃)—.

Each alkenyl moiety either alone or as part of a larger group such asalkenyloxy or alkenylene is a straight or branched chain and ispreferably C₂-C₆alkenyl, more preferably C₂-C₄alkenyl. Each moiety canbe of either the (E)- or (Z)-configuration. Examples include vinyl andallyl. A compound of the present invention comprising an alkenyl moietythus may include, if applicable, either said compound with said alkenylmoiety in its (E)-configuration, said compound with said alkenyl moietyin its (Z)-configuration and mixtures thereof in any ratio.

Each alkynyl moiety either alone or as part of a larger group such asalkynyloxy is a straight or branched chain and is preferablyC₂-C₆alkynyl, more preferably C₂-C₄alkynyl. Examples are ethynyl andpropargyl.

Halogen is fluorine, chlorine, bromine, or iodine, preferably chlorine.In a preferred embodiment, the halogen substituent is chlorine.

Each haloalkyl moiety either alone or as part of a larger group such ashaloalkoxy is an alkyl group substituted by one or more of the same ordifferent halogen atoms. Examples include difluoromethyl,trifluoromethyl, chlorodifluoromethyl and 2,2,2-trifluoro-ethyl.

The term “aryl”, as used herein, refers to a monovalent aromatichydrocarbon radical of 6-14 carbon atoms (C₆-C₁₄) derived by the removalof one hydrogen atom from a single carbon atom of a parent aromatic ringsystem as well as said aryl optionally substituted independently withone or more substituents, typically and preferably with one or twosubstituents as described below. Aryl includes bicyclic radicalscomprising an aromatic ring fused to a saturated, partially unsaturatedring, or aromatic carbocyclic or heterocyclic ring. Aryl groups areoptionally substituted independently with one or more substituents,typically and preferably with one or two substituents, wherein saidsubstituents are independently at each occurrence selected fromC₁-C₄alkyl, halogen, CF₃, OH, C₁-C₃alkoxy, NR₂₀R₂₁, C₆H₅, C₆H₅substituted with halogen, C₁-C₃alkyl, C₁-C₃alkoxy, NR₂₀R₂₁, wherein R₂₀,R₂₁ are independently at each occurrence H, C₁-C₃alkyl. Typical arylgroups include, but are not limited to, radicals derived from benzene(phenyl), substituted phenyls, naphthalene, anthracene, biphenyl,indenyl, indanyl, 1,2-dihydronapthalene, 1,2,3,4-tetrahydronaphthyl andthe like. The term “aryl”, as used herein, preferably refers to phenyloptionally substituted with 1 to 3 R₂₂, wherein R₂₂ is independently ateach occurrence halogen, —OH, C₁-C₃alkyl optionally substituted with oneor two OH, C₁-C₂fluoroalkyl, C₁-C₂alkoxy, C₁-C₂alkoxyC₁-C₃alkyl,C₃-C₆cycloalkyl, —NH₂, NHCH₃ or N(CH₃)₂.

Where a group is said to be optionally substituted, preferably there areoptionally 1-5 substituents, more preferably optionally 1-3substituents, again more preferably optionally 1 or 2 substituents.Where a group is said to be optionally substituted, and where there aremore than one substituents for said optional substitution of said group,said more than one substituents can either be the same or different.

The term “nucleobase”, as used herein, and abbreviated as B_(x), refersto unmodified or naturally occurring nucleobases as well as modified ornon-naturally occurring nucleobases and synthetic mimetics thereof. Anucleobase is any heterocyclic base that contains one or more atoms orgroups of atoms capable of hydrogen bonding to a heterocyclic base of anucleic acid.

In one embodiment, the nucleobase is a purine base or a pyrimidine base,wherein preferably said purine base is purine or substituted purine, andsaid pyrimidine base is pyrimidine or substituted pyrimidine. Morepreferably, the nucleobase is (i) adenine (A), (ii) cytosine (C), (iii)5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U), or (vi)5-methyluracil (MeU), or to a derivative of (i), (ii), (iii), (iv), (v)or (vi). The terms “derivative of (i), (ii), (iii), (iv), (v) or (vi),and “nucleobase derivative” are used herein interchangeably. Derivativesof (i), (ii), (iii), (iv), (v) or (vi), and nucleobase derivatives,respectively, are known to the skilled person in the art and aredescribed, for example, in Sharma V. K. et al., Med. Chem. Commun.,2014, 5, 1454-1471, and include without limitation 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, alkyl adenine, such as6-methyl adenine, 2-propyl adenine, alkyl guanine, such as 6-methylguanine, 2-propyl guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halo uracil, 5-halo cytosine, alkynyl pyrimidinebases, such as 5-propynyl (—C═C—CH₃) uracil, 5-propynyl (—C═C—CH₃)cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, pseudo-uracil,4-thiouracil; 8-substituted purine bases, such as 8-halo-, 8-amino-,8-thiol-, 8-thioalkyl-, 8-hydroxyl-adenine or guanine, 5-substitutedpyrimidine bases, such as 5-halo-, particularly 5-bromo-,5-trifluoromethyl-uracil or -cytosine; 7-methylguanine, 7-methyladenine,2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine,7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine,hydrophobic bases, promiscuous bases, size-expanded bases, orfluorinated bases. In certain embodiments, the nucleobase includeswithout limitation tricyclic pyrimidines, such as1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one or9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). The term“nucleobase derivative” also includes those in which the purine orpyrimidine base is replaced by other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine or 2-pyridone.Further nucleobases of the invention include without limitation thoseknown to skilled artisan (e.g. U.S. Pat. No. 3,687,808; Swayze et al.,The Medicinal Chemistry of Oligonucleotides, in Antisense a DrugTechnology, Chapter 6, pp. 143-182 (Crooke, S. T., ed., 2008); TheConcise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, pp. 858-859; Englisch et al.,Angewandte Chemie, International Edition, 1991, Vol. 30 (6), pp.613-623; Sanghvi, Y. S., Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, pp. 273-302).

Preferred nucleobase derivatives include methylated adenine, guanine,uracil and cytosine and nucleobase derivatives, preferably of (i), (ii),(iii) or (iv), wherein the respective amino groups, preferably theexocyclic amino groups, are protected by acyl protecting groups ordialkylformamidino, preferably dimethylformamidino (DMF), and furtherinclude nucleobase derivatives such as 2-fluorouracil, 2-fluorocytosine,5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine andpyrimidine analogs such as pseudoisocytosine and pseudouracil.

In a further preferred embodiment, said nucleobase derivative isselected from methylated adenine, methylated guanine, methylated uraciland methylated cytosine, and from a nucleobase derivative of (i), (ii),(iii) or (iv), wherein the respective amino groups, preferably theexocyclic amino groups, are protected by a protecting group.

In a further preferred embodiment, said nucleobase derivative isselected from methylated adenine, methylated guanine, methylated uraciland methylated cytosine, and from a nucleobase derivative of (i), (ii),(iii) or (iv), wherein the respective amino groups, preferably theexocyclic amino groups, are protected by acyl protecting groups ordialkylformamidino, preferably dimethylformamidino (DMF).

In a further preferred embodiment, said nucleobase derivative isselected from a nucleobase derivative of (i), (ii), (iii) or (iv),wherein the respective amino groups, preferably the exocyclic aminogroups, are protected by a protecting group.

In a further preferred embodiment, said nucleobase derivative is anucleobase derivative of (i), (ii), (iii) or (iv), wherein the exocyclicamino groups, are protected by acyl protecting groups ordialkylformamidino, preferably dimethylformamidino (DMF).

In a further very preferred embodiment, said acyl protecting group ofsaid exocyclic amino group of said nucleobase derivative of (i), (ii),(iii) or (iv) is —C(O)—R₁₁, wherein independently of each other R₁₁ isselected from C₁-C₁₀alkyl, C₆-C₁₀aryl, C₆-C₁₀arylC₁-C₁₀alkylene, orC₆-C₁₀aryloxyC₁-C₁₀alkylene and wherein said dialkylformamidinoprotecting group is ═C(H)—NR₁₂R₁₃, wherein R₁₂ and R₁₃ are independentlyof each other selected from C₁-C₄alkyl.

In a further very preferred embodiment, said acyl protecting group ofsaid exocyclic amino group of said nucleobase derivative of (i), (ii),(iii) or (iv) is —C(O)—R₁₄, wherein independently of each other R₁₄ isselected from C₁-C₄alkyl; phenyl; phenyl substituted with halogen,C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₄alkoxy; benzyl; benzyl substitutedwith halogen, C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₄alkoxy; orphenyloxyC₁-C₂alkylene optionally substituted with halogen, C₁-C₆alkyl,C₁-C₄alkoxy; and wherein said dialkylformamidino protecting group is═C(H)—NR₁₂R₁₃, wherein R₁₂ and R₁₃ are independently of each otherselected from C₁-C₄alkyl.

In a further very preferred embodiment, said acyl protecting group ofsaid exocyclic amino group of said nucleobase derivative of (i), (ii),(iii) or (iv) is —C(O)—R₁₅, wherein independently of each other R₁₅ isselected from C₁-C₄alkyl; phenyl; phenyl substituted with halogen,C₁-C₄alkyl, C₅-C₆cycloalkyl, C₁-C₄alkoxy; benzyl; benzyl substitutedwith halogen, C₁-C₄alkyl, C₁-C₄alkoxy; or phenyloxymethylene (CH₂—OC₆H₅)wherein the phenyl is optionally substituted with halogen, C₁-C₄alkyl,C₅-C₆cycloalkyl, C₁-C₄alkoxy; and wherein said dialkylformamidinoprotecting group is ═C(H)—NR₁₂R₁₃, wherein R₁₂ and R₁₃ are independentlyof each other selected from C₁-C₄alkyl.

In a further very preferred embodiment, said acyl protecting group ofsaid exocyclic amino group of said nucleobase derivative of (i), (ii),(iii) or (iv) is —C(O)—R₁₆, wherein independently of each other R₁₆ isselected from C₁-C₃alkyl; phenyl; phenyl substituted with C₁-C₃alkyl,methoxy; benzyl; benzyl substituted with C₁-C₃alkyl, methoxy; orphenyloxymethylene (CH₂—OC₆H₅) wherein the C₆H₅ is optionallysubstituted with C₁-C₃alkyl, methoxy; and wherein saiddialkylformamidino protecting group is ═C(H)—NR₁₂R₁₃, wherein Rig andR₁₃ are independently of each other selected from C₁-C₄alkyl.

In a further very preferred embodiment, said acyl protecting group ofsaid exocyclic amino group of said nucleobase derivative of (i), (ii),(iii) or (iv) is —C(O)—R₁₇, wherein independently of each other R₁₇ isselected from C₁-C₃alkyl; phenyl; phenyl substituted with C₁-C₃alkyl,methoxy; benzyl; benzyl substituted with C₁-C₃alkyl, methoxy; orphenyloxymethylene (CH₂—OC₆H₅) wherein the C₆H₅ is optionallysubstituted with C₁-C₃alkyl, methoxy; and wherein saiddialkylformamidino protecting group is dimethylformamidino (DMF).

In a further very preferred embodiment, said acyl protecting group ofsaid exocyclic amino group of said nucleobase derivative of (i), (ii),(iii) or (iv) is —C(O)—R₁₈, wherein independently of each other Rig isselected from methyl, iso-propyl, phenyl, benzyl, or phenyloxymethylene(CH₂—OC₆H₅) wherein the C₆H₅ is optionally substituted with C₁-C₃alkyl,methoxy; and wherein said dialkylformamidino protecting group isdimethylformamidino (DMF).

In a further very preferred embodiment, said acyl protecting group ofsaid exocyclic amino group of said nucleobase derivative of (i), (ii),(iii) or (iv) is —C(O)—R₁₉, wherein independently of each other Rig isselected from methyl, iso-propyl, phenyl, benzyl, or phenyloxymethylene(CH₂—OC₆H₅) wherein the C₆H₅ is optionally substituted with methyl,iso-propyl; and wherein said dialkylformamidino protecting group isdimethylformamidino (DMF).

The term “dialkylformamidino”, as used herein refers to ═C(H)—NR₁₂R₁₃,wherein R₁₂ and R₁₃ are independently of each other selected fromC₁-C₄alkyl. In preferred embodiments, said dialkylformamidino is aprotecting group of said exocyclic amino group of said nucleobasederivative of (i), (ii), (iii) or (iv). The resulting compounds may beof either the (E)- or (Z)-configuration and both forms, and mixturesthereof in any ratio, should be included within the scope of the presentinvention. In a preferred embodiment the inventive compounds comprisethe dialkylformamidino, preferably dimethylformamidino (DMF), in the (Z)configuration.

According to one embodiment, Bx is selected from uracil, thymine,cytosine, 5-methylcytosine, adenine and guanine. Preferably, Bx isselected from thymine, 5-methylcytosine, adenine and guanine. Accordingto one embodiment, Bx is an aromatic heterocyclic moiety capable offorming base pairs when incorporated into DNA or RNA oligomers in lieuof the bases uracil, thymine, cytosine, 5-methylcytosine, adenine andguanine.

The term “nucleosidic linkage group”, as used herein, refers to anylinkage group known in the art that is able to link, preferably links,said inventive compound of formula (IV), (V) or (VI) to a furthercompound, preferably to a nucleosidic compound including a furtherinventive compound of formula (IV), (V) or (VI), within the oligomers inaccordance with the present invention. Representative patents that teachsuch possible linkage groups are without limitation U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437;5,677,439; 5,646,269 and 5,792,608. Said further compound is selectedfrom a nucleosidic compound or a non-nucleosidic compound. Saidnucleosidic compound includes without limitation, and is typically andpreferably selected from, at least one (i) nucleoside, (ii) nucleotide,(iii) oligonucleotide or (iv) modifications of (i), (ii) or (iii). Saidnon-nucleosidic compound includes, and is typically and preferablyselected from, a peptide, protein, silicate compounds or even a solidsupport. The solid support includes without a limitation surfaces,beads, glass supports, polymers or resins. In a preferred embodiment,the glass is controlled-pore glass, preferably with 500 Å, 1000 Å or2000 Å pores. The beads include without limitation glass beads,preferably controlled-pore glass, or magnetic beads. The polymerincludes without limitation polystyrenes including for exampledivinylbenzene, styrene, and chloromethylstyrene. In a preferredembodiment, the solid support are highly cross-linked polystyrene beads.

The term “nucleosidic linkage group” includes phosphorus linkage groupsand non-phosphorus linkage groups. Non-phosphorus linkage groups do notcontain a phosphorus atom and examples of non-phosphorus linkage groupsinclude, and is typically and preferably selected from, alkyl, aryl,preferably, phenyl, benzyl, or benzoyl, cycloalkyl, alkylenearyl,alkylenediaryl, alkoxy, alkoxyalkylene, alkylsulfonyl, alkyne, ether,each independently of each other optionally substituted with cyano,nitro, halogen; carboxyl, amide, amine, amino, imine, thiol, sulfide,sulfoxide, sulfone, sulfamate, sulfonate, sulfonamide, siloxane ormixtures thereof. In a preferred embodiment, the non-phosphorus linkagegroup is amino propyl, long chain alkyl amine group, inyl, acetylamide,aminomethyl, formacetal, thioformacetal, thioformacetyl, riboacetyl,methyleneimino, methylenehydrazino or a neutral non-ionic nucleosidelinkage group, such as amide-3 (3′-CH₂—C(═O)—N(H)-5′) or amide-4(3′-CH₂—N(H)—C(═O)-5′). In a preferred embodiment, the non-phosphoruslinkage group includes a compound selected from alkyl, aryl, preferablyphenyl, benzyl, or benzoyl, cycloalkyl, alkylenearyl, alkylenediaryl,alkoxy, alkoxyalkylene, alkylsulfonyl, alkyne, or ether, wherein thecompound includes C₁-C₉, C₁-C₆, or C₁-C₄.

In a preferred embodiment, said nucleosidic linkage group is aphosphorus linkage group, and said phosphorus linkage group refers to amoiety comprising a phosphorus atom in the P^(III) or P^(V) valencestate represented by formula (XI):

wherein

W represents O, S, Se or an electron pair; preferably W represents O orS;

R₁₀ is H, halogen, OH, OR₅, NR₆R₇, SH, SR₈, C₁-C₆alkyl, C₁-C₆haloalkyl,C₁-C₆alkoxy, C₁-C₆haloalkoxy, C₁-C₆aminoalkyl; wherein R₅ is C₁-C₉alkyl,C₁-C₆alkoxy, each independently of each other optionally substitutedwith cyano, nitro, halogen, —NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl,C₁-C₃alkylsulfonyl; aryl, C₁-C₆alkylenearyl, C₁-C₆alkylenediaryl, eachindependently of each other optionally substituted with cyano, nitro,halogen, C₁-C₄alkoxy, C₁-C₄haloalkyl, C₁-C₄haloalkoxy, NHC(O)C₁-C₃alkyl,NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; acetyl; a hydroxyl protectinggroup; wherein R₆ and R₇ are independently of each other hydrogen,C₁-C₉alkyl optionally substituted with cyano, nitro, halogen,C₂-C₆alkenyl, C₃-C₆cycloalkyl, C₁-C₃alkoxy; aryl optionally substitutedwith cyano, nitro, halogen, C₁-C₃alkyl, C₁-C₃alkoxy; an amino protectinggroup; or together with the nitrogen atom to which they are attachedform a heterocyclic ring, wherein preferably said heterocyclic ring isselected from pyrollidinyl, piperidinyl, morpholinyl, piperazinyl andhomopiperazine, wherein said heterocyclic ring is optionally substitutedwith C₁-C₃ alkyl; and wherein R₈ is a thiol protecting group; andwherein each of the wavy lines indicates the attachment of saidphosphorus linkage group of formula (XI) to a further compound,preferably to a nucleosidic compound including a further inventivecompound of formula (IV), (V) or (VI), within the oligomers inaccordance with the present invention. When W represents O, S or Se thensaid P atom within said phosphorus moiety is in its P^(V) valence state.When W represents an electron pair then said P atom within saidphosphorus moiety is in its P^(III) valence. The moiety of formula (XI)includes any possible stereoisomer. Further included in said moietiesrepresented by formula (XI) are salts thereof, wherein typically andpreferably said salts are formed upon treatment with inorganic bases oramines, and are typically and preferably salts derived from reactionwith the OH or SH groups being (independently of each other) said R₁₀.Preferred inorganic bases or amines leading to said salt formation withthe OH or SH groups are well known in the art and are typically andpreferably trimethylamine, diethylamine, methylamine or ammoniumhydroxide. These phosphorus moieties included in the present inventionare, if appropriate, also abbreviated as “O⁻HB⁺”, wherein said HB⁺refers to the counter cation formed.

In a preferred embodiment, in the phosphorus linkage group of formula(XI), R₆ and R₇ are independently of each other hydrogen, C₁-C₆alkyloptionally substituted with cyano, nitro, halogen, C₂-C₆alkenyl; aryloptionally substituted with cyano, nitro, halogen, C₁-C₃ alkyl; or anamino protecting group.

In a preferred embodiment, in the phosphorus linkage group of formula(XI), W represents O or S; R₁₀ is H, OH, OR₅, NR₆R₇, C₁-C₆alkyl,C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,C₁-C₆aminoalkyl; wherein R₅ is C₁-C₉alkyl, C₁-C₆alkoxy, eachindependently of each other optionally substituted with cyano, nitro,halogen, —NHC(O)C₁-C₃alkyl, —NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl;aryl, C₁-C₆alkylenearyl, C₁-C₆alkylenediaryl each independently of eachother optionally substituted with cyano, nitro, halogen, C₁-C₄alkoxy,C₁-C₄haloalkyl, C₁-C₄haloalkoxy, —NHC(O)C₁-C₃alkyl,NHC(O)C₁-C₃haloalkyl, C₁-C₃alkylsulfonyl; acetyl; or a hydroxylprotecting group; wherein R₆ and R₇ are independently of each otherhydrogen, C₁-C₆alkyl optionally substituted with cyano, nitro, halogen,C₂-C₆alkenyl, C₃-C₆cycloalkyl, C₁-C₃alkoxy; aryl optionally substitutedwith cyano, nitro, halogen, C₁-C₃ alkyl, C₁-C₃alkoxy; an aminoprotecting group; and wherein R₈ is a thiol protecting group.

In further preferred embodiment, said nucleosidic linkage group is aphosphorus linkage group, and said phosphorus linkage group is selectedfrom a phosphodiester linkage group, a phosphotriester linkage group, aphosphorothioate linkage group, a phosphorodithioate linkage group, aphosphonate linkage group, preferably a H-phosphonate linkage group or amethylphosphonate linkage group; a phosphonothioate linkage group,preferably a H-phosphonothioate linkage group, a methyl phosphonothioatelinkage group; a phosphinate linkage group, a phosphorthioamidatelinkage, a phosphoramidate linkage group, or a phosphite linkage group.In another very preferred embodiment, said nucleosidic linkage group isa phosphorus linkage group, and wherein said phosphorus linkage group isselected from a phosphodiester linkage group, a phosphotriester linkagegroup, a phosphorothioate linkage group, or a phosphonate linkage group,wherein the phosphonate is preferably a H-phosphonate linkage group ormethylphosphonate linkage group.

In another very preferred embodiment, said nucleosidic linkage group isa phosphorus linkage group, and wherein said phosphorus linkage group isa phosphodiester linkage group. In another very preferred embodiment,said nucleosidic linkage group is a phosphorus linkage group, andwherein said phosphorus linkage group is a phosphorothioate linkagegroup.

In a preferred embodiment, the phosphorus linkage group is selected froman alkyl phosphodiester linkage group, an alkylene phosphodiesterlinkage group, a thionoalkyl phosphodiester linkage group or anaminoalkyl phosphodiester linkage group, an alkyl phosphotriesterlinkage group, an alkylene phosphotriester linkage group, a thionoalkylphosphotriester linkage group or an aminoalkyl phosphotriester linkagegroup, an alkyl phosphonate linkage group, an alkylene phosphonatelinkage group, an aminoalkyl phosphonate linkage group, a thionoalkylphosphonate linkage group or a chiral phosphonate linkage group. Morepreferably, said nucleosidic linkage group is a phosphorus linkagegroup, and wherein said phosphorus linkage group is a is aphosphodiester linkage group —O—P(═O)(OH)O— or —O—P(═O)(O⁻)O— with [HB⁺]as counterion, a phosphorothioate —O—P(═S)(OH)O— or —O—P(═S)(O⁻)O— with[HB⁺] as counterion, a methylphosphonate —O—P(═O)(CH₃)O—. Various salts,mixed salts and free acid forms of the phosphorus linkage group areincluded.

In a further embodiment, said nucleosidic linkage group links anucleoside, nucleotide or oligonucleotide with a further nucleoside,nucleotide or oligonucleotide.

The wavy line within formulas (I) and (IV) symbolizing the bond betweenthe Bx and and the bicyclic core of the inventive compounds indicatesthat any spatial orientation of the nucleobase Bx are covered by formula(I) or (IV). That means that formulas (I) and (IV) cover either thealpha or the beta conformation or any mixture of alpha and beta anomersof the inventive compounds.

As used herein, the term “nucleoside” refers to a compound comprising anucleobase and a sugar covalently linked to said nucleobase. The term“nucleotide”, as used herein, refers to a nucleoside further comprisinga nucleosidic linkage group or phosphorus moiety, wherein saidnucleosidic linkage group or said phosphorus moiety is covalently linkedto the sugar of said nucleoside.

As used herein the term “nucleoside” or “nucleotide” is meant to includeall manner of naturally occurring or modified nucleosides or nucleosidemimetics, or naturally occurring or modified nucleotides or nucleotidemimetics, respectively, that can be incorporated into an oligomer usingnatural or chemical oligomer synthesis. Typically and preferably, theterm “nucleoside”, as used herein, refers to a naturally occurringnucleoside, a modified nucleoside or nucleoside mimetic. Typically andpreferably, the term “nucleotide”, as used herein, refers to a naturallyoccurring nucleotide, a modified nucleotide or nucleotide mimetic.

The term “modified nucleosides” is intended to include modificationsmade to the sugar and/or nucleobase of a nucleoside as known to theskilled person in the art and described herein. The term “modifiednucleotides” is intended to include modifications made to the sugarand/or nucleobase and/or nucleosidic linkage group or phosphorus moietyof a nucleotide as known to the skilled person in the art and describedherein.

The term “nucleoside mimetic” is intended to include those structuresused to replace the sugar and the nucleobase. Examples of nucleosidemimetics include nucleosides wherein the nucleobase is replaced with aphenoxazine moiety (for example the9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one group) and the sugar moietyis replaced a cyclohexenyl or a bicyclo[3.1.0]hexyl moiety. The term“nucleotide mimetic” as used herein is meant to include nucleotides usedto replace the sugar and the nucleosidic linkage group. Examples ofnucleotide mimetics include peptide nucleic acids (PNA) or morpholinos.

The term “nucleoside” or “nucleotide” also includes combinations ofmodifications, such as more than one nucleobase modification, more thanone sugar modification or at least one nucleobase and at least one sugarmodification.

The sugar of the nucleoside or nucleotide includes without limitation amonocyclic, bicyclic or tricyclic ring system, preferably a tricyclic orbicyclic system or a monocyclic ribose or de(s)oxyribose. Modificationsof the sugar further include but are not limited to modifiedstereochemical configurations, at least one substitution of a group orat least one deletion of a group. A modified sugar is typically andpreferably a modified version of the ribosyl moiety as naturallyoccurring in RNA and DNA (i.e. the furanosyl moiety), such as bicyclicsugars, tetrahydropyrans, 2′-modified sugars, 3′-modified sugars,4′-modified sugars, 5′-modified sugars, or 4′-substituted sugars.Examples of suitable sugar modifications are known to the skilled personand include, but are not limited to 2′, 3′ and/or 4′ substitutednucleosides (e.g. 4′-S-modified nucleosides); 2′-O-modified RNAnucleotide residues, such as 2′-O-alkyl or 2′-O-(substituted)alkyl e.g.2′-O-methyl, 2′-O-(2-cyanoethyl), 2′-O-(2-methoxy)ethyl (2′-MOE),2′-O-(2-thiomethyl)ethyl; 2′-O-(haloalkoxy)methyl e.g.2′-O-(2-chloroethoxy)methyl (MCEM), 2′-O-(2,2-dichloroethoxy)methyl(DCEM); 2′-O-alkoxycarbonyl e.g. 2′-O-[2-(methoxycarbonyl)ethyl] (MOCE),2′-O-[2-(N-methylcarbamoyl)ethyl] (MCE),2′-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DMCE), in particular a2′-O-methyl modification or a 2′-O-methoxyethyl (2′-O-MOE); or othermodified sugar moieties, such as morpholino (PMO), cationic morpholino(PMOPlus) or a modified morpholino group, such as PMO-X. The term“PMO-X” refers to a modified morpholino group comprising at least one 3′or 5′ terminal modification, such 3′-fluorescent tag, 3′ quencher (e.g.3′-carboxyfluorescein, 3′-Gene Tools Blue, 3′-lissamine, 3′-dabcyl),3′-affinity tag and functional groups for chemical linkage (e.g.3′-biotin, 3′-primary amine, 3′-disulfide amide, 3′-pyridyl dithio),5′-end modifications (5′-primary amine, 5′-dabcyl), 3′-azide, 3′-alkyne,5′-azide, 5′-alkyne, or as disclosed in WO2011/150408 andUS2012/0065169.

“Bicylic sugar moieties” comprise two interconnected ring systems, e.g.bicyclic nucleosides wherein the sugar moiety has a 2′-O—CH(alkyl)-4′ or2′-O—CH2-4′ group, locked nucleic acid (LNA), xylo-LNA, alpha-L-LNA,beta-D-LNA, cEt (2′-O,4′-C constrained ethyl) LNA, cMOEt (2′-O,4′-Cconstrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA),hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA(p-RNA), or 3′-deoxypyranosyl-DNA (p-DNA). Alternatively, the sugar ofthe nucleoside or nucleotide includes a tricyclic sugar moiety as, forexample, described in WO 2013/135900 and WO 2014/140348.

The term “oligomer”, as used herein, refers to a compound comprising twoor more monomer subunits linked by nucleosidic linkage groups, whereinat least one of said two or more monomer subunits is a compound of theformula (IV), preferably a compound of the formula (V) or a compound ofthe formula (VI). In a preferred embodiment, the oligomer comprises atleast one compound of formula (IV), (V) or (VI) and at least oneribonucleotide or deoxyribonucleotide. More preferably, the oligomercomprises at least one compound of formula (IV), (V) or (VI) and atleast one deoxyribonucleotide.

The term “monomer subunit”, as used herein, is meant to include allmanner of monomer units that are amenable to oligomer synthesisincluding, and typically and preferably referring to, monomer subunitssuch as α-D-ribonucleosides, β-D-ribonucleosides,α-D-2′-deoxyribnucleosides, β-D-2′-deoxyribnucleosides, naturallyoccurring nucleosides, naturally occurring nucleotides, modifiednucleosides, modified nucleotides, mimetics of nucleosides, mimetics ofnucleotides, and the inventive compounds provided herein including anycompounds of any one of the formulas (I) to (VI).

In a preferred embodiment, the oligomer is an oligonucleotide. The term“oligonucleotide”, as used herein, refers to a compound comprising atleast two nucleosides linked to each other each by a nucleosidic linkagegroup. Thus, the term “oligonucleotide”, as used herein, includes, andtypically and preferably refer to, compounds comprising at least twonucleosides linked by nucleosidic linkage groups, wherein said at leasttwo nucleosides are independently selected from naturally occurringnucleosides, modified nucleosides or nucleoside mimetics. Thus, the term“oligonucleotide”, as used herein, includes compounds comprisingnaturally occurring nucleotides, modified nucleotides or nucleotidemimetics and, thus, the term “oligonucleotide”, as used herein, includesoligonucleotides with modifications made to the sugar and/or nucleobaseand/or nucleosidic linkage group as known to the skilled person in theart and described herein.

The oligomer can be single stranded or double stranded. In oneembodiment, the oligomer is double stranded (i.e. a duplex). In apreferred embodiment, the oligomer is single stranded.

In a preferred embodiment the oligomer is coupled to a non-nucleosidiccompound, preferably a solid support. The solid support is preferablyselected from beads, polymers or resin. In a certain embodiment, theoligomer has a length of up to 40 monomer subunits, preferably up to 30monomer subunits, more preferably up to 30 monomer subunits, again morepreferably up to 20 monomer subunits or up to 15 monomer subunits. In afurther embodiment, said oligomer comprises from 5 to 40 monomericsubunits, preferably from 8 to 30 monomer subunits, more preferably from8 to 25 monomer subunits, again more preferably from 8 to 20 monomersubunits.

In certain embodiments, the oligomer as provided herein is modified bycovalent attachment of one or more terminal groups to the 5′ or 7′terminus of the oligomer. A terminal group can also be attached at anyof the termini of the oligomer.

The term “terminus” refers to the end or terminus of the oligomer,nucleic acid sequence or the compound of formula (IV), (V) or (VI),wherein the integer (3′, 5′ or 7′ etc.) indicates to the carbon atom ofthe sugar included in the nucleoside of the oligomer, nucleic acidsequence or the compound of formula (IV), (V) or (VI). The term “5′terminal group” or “7′ terminal group”, as used herein, refers to agroup located at the 5′ terminus or 7′ terminus, respectively, of thesugar included in the compound of formula (IV), (V) or (VI). Examples ofthe “5′ terminal group” or “7′ terminal group” include withoutlimitation a capping group, diphosphate, triphosphate, label, such as afluorescent label (e.g. fluorescein or rhodamine), dye, reporter groupsuitable for tracking the oligomer, solid support, non-nucleosidicgroup, antibody or conjugate group. Preferably a “5′ terminal group” or“7′ terminal group” is selected from a diphosphate, triphosphate,fluorescent label, dye, reporter group that can track the oligomer,solid support, non-nucleosidic group, antibody or conjugate group.

In certain embodiments, the oligomer as provided herein or the compoundof formula (IV), (V) or (VI) is modified by covalent attachment of oneor more conjugate groups. In general, conjugate groups modify one ormore properties of the compound they are attached to. Such propertiesinclude without limitation, nuclease stability, binding affinity,pharmacodynamics, pharmacokinetics, binding, absorption, cellulardistribution, cellular uptake, delivery, charge and clearance. Conjugategroups are routinely used in the chemical arts and are linked directlyor via an optional linkage group to a parent compound such as anoligomer. The term “conjugate group” includes without limitation, andrefers preferably to intercalators, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, lipophilicmoieties, or coumarins.

The term “nucleic acid” or “nucleic acid sequence”, as interchangeablyused herein, is understood as oligomeric or polymeric moleculecomprising at least two interlinked nucleotides or at least twonucleosides linked by a nucleosidic linkage group. In the context of thepresent invention, the nucleic acid includes ribonucleic acid (RNA) anddeoxyribonucleic acid (DNA) and is preferably selected from naturallyoccurring RNA, naturally occurring DNA, modified DNA, modified RNA,mixtures thereof, such as RNA-DNA hybrids. The modification may comprisethe backbone such as the nucleosidic linkage group and/or the nucleosideand/or the sugar as further described herein. The nucleic acids can besynthesized chemically or enzymatically by polymerases.

The term “natural” or “naturally occurring”, as interchangeably usedherein, refers to compounds that are of natural origin.

The term “stereoisomers” refers to compounds, which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality in which the compounds are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and chemical and biologicalreactivities. Mixtures of diastereomers may be separated under highresolution analytical procedures such as electrophoresis andchromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McRaw-Hiff Dictionary of Chemical Terms (1984),McGraw-Hill Book Company, N.Y. and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994.

As used herein, “T_(m)” (melting temperature) is the temperature atwhich two strands of a duplex nucleic acid separate. The T_(m) is oftenused as a measure of duplex stability of an antisense compound toward acomplementary nucleic acid.

In a first aspect, the present invention provides a compound of formula(I):

wherein one of T₁ and T₂ is OR₁ or OR₂;and the other of T₁ and T₂ is OR₁ or OR₂; whereinR₁ is H or a hydroxyl protecting group, andR₂ is a phosphorus moiety; and whereinBx is a nucleobase.

In a preferred embodiment, said compound of formula (I) of the inventionis a compound of formula (II)

wherein(i) T₁ is OR₁, and T₂ is OR₁ or OR₂; or(ii) T₁ is OR₁ or OR₂, T₂ is OR₁:wherein preferably T₁ is OR₁ or OR₂, T₂ is OR₁.

The compound of formula (II) is an alpha anomer or an alpha anomericmonomer that differs from the beta anomer in the spatial configurationof Bx at the chiral center of the first carbon at the 1′ terminus.

In another preferred embodiment, said compound of formula (I) is acompound of formula (III)

wherein(i) T₁ is OR₁, and T₂ is OR₁ or OR₂; or(ii) T₁ is OR₁ or OR₂, T₂ is OR₁:wherein preferably T₁ is OR₁, and T₂ is OR₁ or OR₂.

The compound of formula (III) is a beta anomer or a beta anomericmonomer that differs from the alpha anomer in the spatial configurationof Bx at the chiral center of the first carbon at the 1′ terminus.

In another preferred embodiment, in the compound of formula (I), saidphosphorus moiety R₂ is selected from a phosphate moiety, aphosphoramidate moiety and a phosphoramidite moiety. In anotherpreferred embodiment, in the compound of formula (II) said phosphorusmoiety R₂ is selected from a phosphate moiety, a phosphoramidate moietyand a phosphoramidite moiety. In another preferred embodiment, in thecompound of formula (III) said phosphorus moiety R₂ is selected from aphosphate moiety, a phosphoramidate moiety and a phosphoramidite moiety.

In another preferred embodiment, in the compound of formula (I), (II) or(III) said Bx is selected from a purine base or pyrimidine base, whereinpreferably Bx is selected from (i) adenine (A), (ii) cytosine (C), (iii)5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U), or (vi)5-methyluracil (MeU), or a derivative of (i), (ii), (iii), (iv), (v) or(vi), and wherein further preferably Bx is selected from uracil,thymine, cytosine, 5-methylcytosine, adenine or guanine. Again morepreferably, in the compound of formula (I), (II) or (III), Bx isselected from thymine, 5-methylcytosine, adenine or guanine.

In another preferred embodiment, the compound of formula (I), (II) or(III) is linked to a non-nucleosidic compound, preferably a solid-phase.

In a preferred embodiment, the compound of formula (I) is selected from:

In a second aspect, the invention provides an oligomer comprising atleast one compound of formula (IV)

wherein independently for each of said at least one compound of formula(IV)one of T₃ or T₄ is a nucleosidic linkage group;the other of T₃ and T₄ is OR₁, OR₂, a 5′ terminal group, a 7′ terminalgroup or a nucleosidic linkage group, wherein R₁ is H or a hydroxylprotecting group, and R₂ is a phosphorus moiety; and Bx is a nucleobase.

In a preferred embodiment, the oligomer of the invention comprises atleast one compound of formula (IV), wherein said compound of formula(IV) is a compound of formula (V):

wherein

-   (i) T₃ is a nucleosidic linkage group, and T₄ is a 7′ terminal    group, OR₁, or OR₂, preferably T₄ is a 7′ terminal group or OR₁; or-   (ii) T₃ is a 5′ terminal group, OR₁, or OR₂, preferably T₃ is a 5′    terminal group or OR₂; and T₄ is a nucleosidic linkage group; or-   (iii) T₃ and T₄ are independently of each other a nucleosidic    linkage group.

In another preferred embodiment, the oligomer of the invention comprisesat least one compound of formula (IV), wherein said compound of formula(IV) is a compound of formula (VI):

wherein

-   (i) T₃ is a nucleosidic linkage group, and T₄ is a 7′ terminal    group, OR₁, or OR₂, preferably T₄ is a 7′ terminal group or OR₂; or-   (ii) T₃ is a 5′ terminal group, OR₁, or OR₂, preferably T₃ is a 5′    terminal group or OR₁; and T₄ is a nucleosidic linkage group; or-   (iii) T₃ and T₄ are independently of each other a nucleosidic    linkage group.

In a preferred embodiment, said oligomer is an oligonucleotide. In afurther preferred embodiment, said oligomer is an oligonucleotide,wherein said compound of formula (IV) is a compound of formula (V). Inanother preferred embodiment, said oligomer is an oligonucleotide,wherein said compound of formula (IV) is a compound of formula (VI). Ina more preferred embodiment, the oligomer comprising said least onecompound of formula (IV), (V) or (VI) is a DNA.

In another embodiment, the inventive oligomer comprising said least onecompound of formula (IV), (V) or (VI) further comprises at least onenucleotide that is different from any one of the compound of formula(IV), (V) or (VI), wherein preferably the at least one differentnucleotide is (i) a nucleotide comprising a monocyclic sugar, i.e. amonocyclic nucleotide, or (ii) a nucleotide comprising a bicyclic sugar,i.e. a bicyclic nucleotide, or (iii) a nucleotide comprising a tricyclicsugar, i.e. a tricyclic nucleotide. Preferably, said at least onenucleotide that is different from the compound of formula (IV), (V) or(VI) is a nucleotide comprising a bicyclic sugar. Preferably, said atleast one nucleotide that is different from the compound of formula(IV), (V) or (VI) is a nucleotide comprising a tricyclic sugar. Morepreferably, said at least one nucleotide that is different from thecompound of formula (IV), (V) or (VI) is a nucleotide comprising amonocyclic sugar.

In another preferred embodiment of the inventive oligomer, said compoundof formula (IV) is a compound of formula (V), and said oligomer furthercomprises at least one nucleotide that is different from the compound offormula (V), wherein preferably the at least one different nucleotide is(i) a nucleotide comprising a monocyclic sugar, i.e. a monocyclicnucleotide, or (ii) a nucleotide comprising a bicyclic sugar, i.e. abicyclic nucleotide, or (iii) a nucleotide comprising a tricyclic sugar,i.e. a tricyclic nucleotide. Preferably, said at least one nucleotidethat is different from the compound of formula (V) is a nucleotidecomprising a bicyclic sugar. Preferably, said at least one nucleotidethat is different from the compound of formula (V) is a nucleotidecomprising a tricyclic sugar. More preferably, said at least onenucleotide that is different from the compound of formula (V) is anucleotide comprising a monocyclic sugar.

In another preferred embodiment of the inventive oligomer, said compoundof formula (IV) is a compound of formula (VI), and said oligomer furthercomprises at least one nucleotide that is different from the compound offormula (VI), wherein preferably the at least one different nucleotideis (i) a nucleotide comprising a monocyclic sugar, i.e. a monocyclicnucleotide, or (ii) a nucleotide comprising a bicyclic sugar, i.e. abicyclic nucleotide, or (iii) a nucleotide comprising a tricyclic sugar,i.e. a tricyclic nucleotide. Preferably, said at least one nucleotidethat is different from the compound of formula (VI) is a nucleotidecomprising a bicyclic sugar. Preferably, said at least one nucleotidethat is different from the compound of formula (VI) is a nucleotidecomprising a tricyclic sugar. More preferably, said at least onenucleotide that is different from the compound of formula (VI) is anucleotide comprising a monocyclic sugar.

In another embodiment, the oligomer comprising the compound of formula(IV), (V) or (VI) further comprises at least two nucleotides that aredifferent from the compound of formula (IV), (V) or (VI), wherein saidat least two different nucleotides are linked to each other by anucleosidic linkage group, wherein each nucleosidic linkage group isindependently of each other selected from a phosphodiester linkagegroup, a phosphotriester linkage group, a phosphorothioate linkagegroup, a phosphorodithioate linkage group, a phosphonate linkage group,a phosphonothioate linkage group, a phosphinate linkage group, aphosphorthioamidate linkage or a phosphoramidate linkage group, andwherein preferably each nucleosidic linkage group is independently ofeach other a phosphodiester linkage group or a phosphorothioate linkagegroup, and wherein further preferably each nucleosidic linkage group isa phosphorothioate linkage group.

In another preferred embodiment of the inventive oligomer, said compoundof formula (IV) is a compound of formula (V), and said oligomer furthercomprises at least two nucleotides that are different from the compoundof formula (V), wherein said at least two different nucleotides arelinked to each other by a nucleosidic linkage group, wherein eachnucleosidic linkage group is independently of each other selected from aphosphodiester linkage group, a phosphotriester linkage group, aphosphorothioate linkage group, a phosphorodithioate linkage group, aphosphonate linkage group, a phosphonothioate linkage group, aphosphinate linkage group, a phosphorthioamidate linkage or aphosphoramidate linkage group, and wherein preferably each nucleosidiclinkage group is independently of each other a phosphodiester linkagegroup or a phosphorothioate linkage group, and wherein furtherpreferably each nucleosidic linkage group is a phosphorothioate linkagegroup.

In another preferred embodiment of the inventive oligomer, said compoundof formula (IV) is a compound of formula (VI), and said oligomer furthercomprises at least two nucleotides that are different from the compoundof formula (VI), wherein said at least two different nucleotides arelinked to each other by a nucleosidic linkage group, wherein eachnucleosidic linkage group is independently of each other selected from aphosphodiester linkage group, a phosphotriester linkage group, aphosphorothioate linkage group, a phosphorodithioate linkage group, aphosphonate linkage group, a phosphonothioate linkage group, aphosphinate linkage group, a phosphorthioamidate linkage or aphosphoramidate linkage group, and wherein preferably each nucleosidiclinkage group is independently of each other a phosphodiester linkagegroup or a phosphorothioate linkage group, and wherein furtherpreferably each nucleosidic linkage group is a phosphorothioate linkagegroup.

In another preferred embodiment, in the oligomer of the invention, Bx isselected from a purine base or pyrimidine base, wherein preferably Bx isselected from (i) adenine (A), (ii) cytosine (C), (iii) 5-methylcytosine(MeC), (iv) guanine (G), (v) uracil (U), or (vi) 5-methyluracil (MeU),or a derivative of (i), (ii), (iii), (iv), (v) or (vi), and whereinfurther preferably Bx is selected from uracil, thymine, cytosine,5-methylcytosine, adenine or guanine. More preferably, in the oligomerof the invention, Bx is selected from thymine, 5-methylcytosine, adenineor guanine.

In another preferred embodiment, in the oligomer of the invention, eachnucleosidic linkage group is independently of each other selected from aphosphodiester linkage group, a phosphotriester linkage group, aphosphorothioate linkage group, a phosphorodithioate linkage group, aphosphonate linkage group, a phosphonothioate linkage group, aphosphinate linkage group, a phosphorthioamidate linkage or aphosphoramidate linkage group, and wherein preferably each nucleosidiclinkage group is independently of each other a phosphodiester linkagegroup or a phosphorothioate linkage group, and wherein furtherpreferably each nucleosidic linkage group is a phosphorothioate linkagegroup.

In another embodiment, the oligomer of the invention comprises 1 to 5,preferably 1 to 4, more preferably 1 to 2, again more preferably 1 to 2,again more preferably exactly one compound of formula (IV), (V) or (VI).In a preferred embodiment of the inventive oligomer, said compound offormula (IV) is a compound of formula (VI), and wherein said oligomercomprises 1 to 5, preferably 1 to 4, more preferably 1 to 2, again morepreferably 1 to 2, again more preferably exactly one compound of formula(VI). It has been found that, in particular, a single incorporation of acompound of formula (VI) within an oligonucleotide, and preferably asingle incorporation of a compound of formula (VI), wherein Bx ismethylcytosine, inside DNA duplexes has a substantial stabilizingeffect. Thus, in a very preferred embodiment of the inventive oligomer,said oligomer comprises exactly one compound of formula (IV), whereinsaid compound of formula (IV) is a compound of formula (VI) and whereinsaid Bx is methylcytosine, and wherein said oligomer is anoligonucleotide and further comprises at least one nucleotide that isdifferent from the compound of formula (VI), wherein preferably the atleast one different nucleotide is a nucleotide comprising a monocyclicsugar.

In another preferred embodiment of the inventive oligomer, said oligomercomprises at least two compounds of formula (IV) and further comprisesat least one nucleotide that is different from the compound of formula(IV), wherein said compound of formula (IV) is a compound of formula(V), and wherein EACH of said compound of formula (V) is linked with its5′ terminus to (i) a 5′ terminus of said at least one nucleotide that isdifferent from the compound of formula (IV) or (ii) a 7′ terminus ofanother compound of formula (V); and wherein said compound of formula(V) is linked with its 7′ terminus to (i) a 3′ terminus of said at leastone nucleotide that is different from the compound of formula (IV) or(ii) a 5′ terminus of another compound of formula (V).

In another embodiment of the inventive oligomer, said oligomer comprisesat least two compounds of formula (IV) and further comprises at leastone nucleotide that is different from the compound of formula (IV),wherein said compound of formula (IV) is a compound of formula (VI), andwherein EACH of said compound of formula (VI) is linked with its 5′terminus to (i) a 3′ terminus of said at least one nucleotide that isdifferent from the compound of formula (IV) or (ii) a 3′ terminus ofanother compound of formula (VI); and wherein EACH of said compound offormula (VI) is linked with its 3′ terminus to (i) a 5′ terminus of saidat least one nucleotide that is different from the compound of formula(IV) or (ii) a 5′ terminus of another compound of formula (VI).

In a preferred embodiment of the inventive oligomer, said oligomerfurther comprises at least two nucleotides that are different from thecompound of formula (IV), and wherein said compound of formula (IV) is acompound of formula (V), and wherein EACH OF said nucleotide that isdifferent from the compound of formula (IV) is linked with its 3′terminus to (i) a 7′ terminus of said compound of formula (V) or (ii) a5′ terminus of another nucleotide that is different from the compound offormula (IV), and wherein EACH of said nucleotide that is different fromthe compound of formula (IV) is linked with its 5′ terminus to (i) a 5′terminus of said compound of formula (V) or (ii) a 3′ terminus ofanother nucleotide that is different from the compound of formula (IV).

In a preferred embodiment of the inventive oligomer, said oligomerfurther comprises at least two nucleotides that are different from thecompound of formula (IV), and wherein said compound of formula (IV) is acompound of formula (V), and wherein EACH of said nucleotide that isdifferent from the compound of formula (IV) is linked with its 3′terminus to (i) a 7′ terminus of said compound of formula (V) or (ii) a5′ terminus of another nucleotide that is different from the compound offormula (IV), and wherein EACH of said nucleotide that is different fromthe compound of formula (IV) is linked with its 5′ terminus to (i) a 5′terminus of said compound of formula (V) or (ii) a 3′ terminus ofanother nucleotide that is different from the compound of formula (IV).

In a preferred embodiment of the inventive oligomer, said oligomerfurther comprises at least two nucleotides that are different from thecompound of formula (IV), and wherein said compound of formula (IV) is acompound of formula (V), and wherein each of the at least one compoundof formula (V) is linked with its 5′ terminus and with its 7′ terminusto said nucleotide that is different from the compound of formula (IV).

In a preferred embodiment of the inventive oligomer, said oligomerfurther comprises at least two nucleotides that are different from thecompound of formula (IV), and wherein said compound of formula (IV) is acompound of formula (V), and wherein EACH of said nucleotide that isdifferent from the compound of formula (IV) is linked with its 3′terminus to (i) a 7′ terminus of said compound of formula (V) or (ii) a5′ terminus of another nucleotide that is different from the compound offormula (IV), and wherein EACH of said nucleotide that is different fromthe compound of formula (IV) is linked with its 5′ terminus to (i) a 5′terminus of said compound of formula (V) or (ii) a 3′ terminus ofanother nucleotide that is different from the compound of formula (IV).

In a preferred embodiment of the inventive oligomer, said oligomerfurther comprises at least two nucleotides that are different from thecompound of formula (IV), and wherein said compound of formula (IV) is acompound of formula (VI), and wherein each of the at least one compoundof formula (VI) is linked with its 5′ terminus and with its 7′ terminusto said nucleotide that is different from the compound of formula (IV).

In another preferred embodiment of the inventive oligomer, said oligomercomprises at least two compounds of formula (IV) and further comprisesat least two nucleotides that are different from the compound of formula(IV), and wherein said compound of formula (IV) is a compound of formula(V), and wherein each of the at least one compound of formula (V) islinked with its 7′ terminus to a 3′ terminus of the nucleotide that isdifferent from the compound of formula (IV); and with its 5′ terminus toa 5′ terminus of a nucleotide that is different from the compound offormula (IV).

In another preferred embodiment of the inventive oligomer, said oligomercomprises at least two compounds of formula (IV) and further comprisesat least two nucleotides that are different from the compound of formula(IV), and wherein said compound of formula (IV) is a compound of formula(VI), and wherein each of the at least one compound of formula (VI) islinked with its 5′ terminus to a 3′ terminus of the nucleotide that isdifferent from the compound of formula (IV); and with its 7′ terminus toa 5′ terminus of a nucleotide that is different from the compound offormula (IV).

In a preferred embodiment of the inventive oligomer, said oligomercomprises at least one compound of formula (IV), wherein said compoundis a compound of formula (VI), wherein each of the at least one compoundof formula (VI) is linked with its 5′ terminus and with its 7′ terminusto a nucleotide that is different from the compound of formula (IV), andwherein Bx is cytosine or 5-methylcytosine, preferably 5-methylcytosine.

In a further preferred embodiment of the inventive oligomer, saidoligomer comprises exactly one compound of formula (IV), wherein saidcompound is a compound of formula (VI), wherein said compound of formula(VI) is linked with its 5′ terminus and with its 7′ terminus to anucleotide that is different from the compound of formula (IV), andwherein Bx is cytosine or 5-methylcytosine, preferably 5-methylcytosine.

In another preferred embodiment, the oligomer of the invention comprises1 to 5, preferably 1 to 4, more preferably 1 to 2, again more preferably1 to 2, again more preferably exactly one compound of formula (IV), (V)or (VI), preferably of formula (VI), wherein Bx is a pyrimidine base,more preferably cytosine or 5-methylcytosine, again more preferably5-methylcytosine. In a more preferred embodiment, the oligomer of theinvention comprises exactly one compound of formula (VI), wherein Bx iscytosine or 5-methylcytosine, preferably 5-methylcytosine. Incorporationof exactly one or only few compounds of formula (IV), (V) or (VI),preferably of formula (VI), wherein Bx is a pyrimidine bases, leads onlyto a small destabilization or even stabilizes duplex formation witholigomers of the invention. Especially, when the nucleobase is acytosine or cytosine derivative, preferably 5-methylcytosine,significantly stabilize duplexes of the oligomer of the inventionincluding compounds of formula (IV), (V) or (VI), preferably of formula(VI), with complementary DNA. This stabilizing effect is more pronouncedfor the 5-methyl cytosine nucleosides. In another embodiment, theoligomer of the invention comprises 1 to 5, preferably 1 to 4, morepreferably 1 to 2, again more preferably 1 to 2, again more preferablyexactly one compound of formula (IV), (V) or (VI), preferably of formula(VI), wherein Bx is a purine base. The nucleobase purine stabilizesduplexes of the oligomers of the invention including compounds offormula (IV), (V) or (VI), preferably of formula (VI), withcomplementary RNA.

In another preferred embodiment, the oligomer of the invention comprisesor preferably consists of at least two contiguous compounds of formula(IV), wherein each of the contiguous compounds of formula (IV) isindependently linked to the adjacent contiguous compound of formula (IV)by the nucleosidic linkage group, wherein the nucleosidic linkage grouplinks a 5′ terminus and a 7′ terminus of two contiguous compounds offormula (IV). In another preferred embodiment, in the oligomer of theinvention comprises or preferably consists of at least two contiguouscompounds of formula (V), wherein each of the contiguous compounds offormula (V) is independently linked to the adjacent contiguous compoundof formula (V) by the nucleosidic linkage group, wherein the nucleosidiclinkage group links a 5′ terminus and a 7′ terminus of two contiguouscompounds of formula (V). In another preferred embodiment, in theoligomer of the invention comprises or preferably consists of at leasttwo contiguous compounds of formula (VI), wherein each of the contiguouscompounds of formula (VI) is independently linked to the adjacentcontiguous compound of formula (VI) by the nucleosidic linkage group,wherein the nucleosidic linkage group links a 5′ terminus and a 7′terminus of two contiguous compounds of formula (VI).

In a further preferred embodiment, the oligomer of the inventioncomprises or preferably consists of 10 to 40 contiguous compounds offormula (IV), preferably 10 to 30 contiguous compounds of formula (IV),more preferably 10 to 25 contiguous compounds of formula (IV), againmore preferably 10 to 20 contiguous compounds of formula (IV) or 10 to15 contiguous compounds of formula (IV). In a further preferredembodiment of the inventive oligomer, said at least one compound offormula (IV) is a compound of formula (V), and wherein said oligomercomprises or preferably consists of 10 to 40 contiguous compounds offormula (V), preferably 10 to 30 contiguous compounds of formula (V),more preferably 10 to 25 contiguous compounds of formula (V), again morepreferably 10 to 20 contiguous compounds of formula (V), and again morepreferably 10 to 15 contiguous compounds of formula (V). In a furtherpreferred embodiment of the inventive oligomer, said at least onecompound of formula (IV) is a compound of formula (VI), and wherein saidoligomer comprises or preferably consists of 10 to 40 contiguouscompounds of formula (VI), preferably 10 to 30 contiguous compounds offormula (VI), more preferably 10 to 25 contiguous compounds of formula(VI), again more preferably 10 to 20 contiguous compounds of formula(VI), and again more preferably 10 to 15 contiguous compounds of formula(VI).

In a further preferred embodiment of the inventive oligomer, said atleast one compound of formula (IV) is a compound of formula (V), andwherein said oligomer comprises or preferably consists of 10 to 40contiguous compounds of formula (V), preferably 10 to 30 contiguouscompounds of formula (V), more preferably 10 to 25 contiguous compoundsof formula (V), again more preferably 10 to 20 contiguous compounds offormula (V), and again more preferably 10 to 15 contiguous compounds offormula (V) and wherein each of the contiguous compounds of formula (V)is independently linked to the adjacent contiguous compound of formula(V) by the nucleosidic linkage group, wherein the nucleosidic linkagegroup links a 5′ terminus and a 7′ terminus of two contiguous compoundsof formula (V), and wherein said nucleosidic linkage group is aphosphorus linkage group, and wherein said phosphorus linkage group isselected from a phosphodiester linkage group, a phosphotriester linkagegroup and a phosphorothioate linkage group, and wherein preferably saidphosphorus linkage group is a phosphodiester linkage group or aphosphorothioate linkage group.

In a further preferred embodiment, the oligomer of the inventioncomprises or preferably consists of at least one nucleic acid sequence,wherein said nucleic acid sequence comprises said at least one compoundof formula (IV), and wherein said nucleic acid sequence is selected fromSEQ ID NO: 1 to 24, preferably SEQ ID NO: 24. In a further preferredembodiment, the oligomer of the invention comprises or preferablyconsists of at least one nucleic acid sequence, wherein said nucleicacid sequence comprises said at least one compound of formula (V), andwherein said nucleic acid sequence is selected from SEQ ID NO: 16 to 24,preferably SEQ ID NO: 21 to 24, more preferably SEQ ID NO: 24. In afurther preferred embodiment, the oligomer of the invention comprises orpreferably consists of at least one nucleic acid sequence, wherein saidnucleic acid sequence comprises said at least one compound of formula(VI), and wherein said nucleic acid sequence is selected from SEQ ID NO:1 to 15, preferably SEQ ID NO: 13-15. In a more preferred embodiment,the oligomer of the invention consists of a nucleic acid sequenceselected from SEQ ID NO: 13 to 15 or 21 to 24, preferably SEQ ID NO: 24.In another preferred embodiment, said oligomer is the nucleic acidsequence selected from SEQ ID NO: 13 to 15 or 21 to 24, and whereinpreferably said oligomer is SEQ ID NO: 24.

In a further preferred embodiment of the inventive oligomer, saidoligomer comprises a nucleic acid sequence, wherein said nucleic acidsequence consists of at least two contiguous compounds of formula (IV),wherein said nucleic acid sequence is flanked on its 5′ terminus or its7′ terminus by at least one nucleotide or nucleoside that is differentfrom the compound of formula (IV). In a further preferred embodiment ofthe inventive oligomer, said oligomer comprises a nucleic acid sequence,wherein said nucleic acid sequence consists of at least two contiguouscompounds of formula (V), wherein said nucleic acid sequence is flankedon its 5′ terminus or its 7′ terminus by at least one nucleotide ornucleoside that is different from the compound of formula (V),preferably different from the compound of formula (V) or (VI), andfurther preferably different from the compound of formula (IV). In afurther preferred embodiment of the inventive oligomer, said, oligomercomprises a nucleic acid sequence, wherein said nucleic acid sequenceconsists of at least two contiguous compounds of formula (VI), whereinsaid nucleic acid sequence is flanked on its 5′ terminus or its 7′terminus by at least one nucleotide or nucleoside that is different fromthe compound of formula (VI), preferably different from the compound offormula (V) or (VI), and further preferably different from the compoundof formula (IV).

In a further preferred embodiment of the inventive oligomer, saidoligomer comprises a nucleic acid sequence, wherein said nucleic acidsequence consists of at least two contiguous compounds of formula (IV),wherein said nucleic acid sequence is flanked on its 5′ terminus and its7′ terminus by at least one nucleotide or nucleoside that is differentfrom the compound of formula (IV). In a further preferred embodiment ofthe inventive oligomer, said oligomer comprises a nucleic acid sequence,wherein said nucleic acid sequence consists of at least two contiguouscompounds of formula (V), wherein said nucleic acid sequence is flankedon its 5′ terminus and its 7′ terminus by at least one nucleotide ornucleoside that is different from the compound of formula (V),preferably different from the compound of formula (V) or (VI), andfurther preferably different from the compound of formula (IV). In afurther preferred embodiment of the inventive oligomer, said oligomercomprises a nucleic acid sequence, wherein said nucleic acid sequenceconsists of at least two contiguous compounds of formula (VI), whereinsaid nucleic acid sequence is flanked on its 5′ terminus and its 7′terminus by at least one nucleotide or nucleoside that is different fromthe compound of formula (VI), preferably different from the compound offormula (V) or (VI), and further preferably different from the compoundof formula (IV).

In a further preferred embodiment of the inventive oligomer, saidoligomer comprises a nucleic acid sequence, wherein said nucleic acidsequence consists of at least two contiguous compounds of formula (V),wherein said nucleic acid sequence is flanked on its 5′ terminus or its7′ terminus by at least one nucleotide or nucleoside that is differentfrom the compound of formula (V), preferably different from the compoundof formula (V) or (VI), and further preferably different from thecompound of formula (IV), wherein the 5′ terminus of said nucleic acidsequence is linked to a 5′ terminus of the nucleotide that is differentfrom the compound of formula (V), preferably different from the compoundof formula (V) or (VI), and further preferably different from thecompound of formula (IV); or wherein the 7′ terminus of said nucleicacid sequence is linked to a 3′ terminus of the nucleotide or nucleosidethat is different from the compound of formula (V), preferably differentfrom the compound of formula (V) or (VI), and further preferablydifferent from the compound of formula (IV).

In a further preferred embodiment of the inventive oligomer, saidoligomer comprises a nucleic acid sequence, wherein said nucleic acidsequence consists of at least two contiguous compounds of formula (V),wherein said nucleic acid sequence is flanked on its 5′ terminus and onits 7′ terminus by at least one nucleotide or nucleoside that isdifferent from the compound of formula (V), preferably different fromthe compound of formula (V) or (VI), and further preferably differentfrom the compound of formula (IV), wherein the 5′ terminus of saidnucleic acid sequence is linked to a 5′ terminus of the nucleotide ornucleoside that is different from the compound of formula (V),preferably different from the compound of formula (V) or (VI), andfurther preferably different from the compound of formula (IV); andwherein the 7′ terminus of said nucleic acid sequence is linked to a 3′terminus of the nucleotide or nucleoside that is different from thecompound of formula (V), preferably different from the compound offormula (V) or (VI), and further preferably different from the compoundof formula (IV).

In a further preferred embodiment of the inventive oligomer, saidoligomer comprises a nucleic acid sequence, wherein said nucleic acidsequence consists of at least two contiguous compounds of formula (VI),wherein said nucleic acid sequence is flanked on its 5′ terminus or onits 7′ terminus by at least one nucleotide or nucleoside t that isdifferent from the compound of formula (VI), preferably different fromthe compound of formula (V) or (VI), and further preferably differentfrom the compound of formula (IV), wherein the 5′ terminus of saidnucleic acid sequence is linked to a 3′ terminus of the nucleotide ornucleoside that is different from the compound of formula (VI),preferably different from the compound of formula (V) or (VI), andfurther preferably different from the compound of formula (IV); orwherein the 7′ terminus of said nucleic acid sequence is linked to a 5′terminus of the nucleotide or nucleoside that is different from thecompound of formula (VI), preferably different from the compound offormula (V) or (VI), and further preferably different from the compoundof formula (IV).

In a further preferred embodiment of the inventive oligomer, saidoligomer comprises a nucleic acid sequence, wherein said nucleic acidsequence consists of at least two contiguous compounds of formula (VI),wherein said nucleic acid sequence is flanked on its 5′ terminus and onits 7′ terminus each by at least one nucleotide or nucleoside that isdifferent from the compound of formula (VI), preferably different fromthe compound of formula (V) or (VI), and further preferably differentfrom the compound of formula (IV), wherein the 5′ terminus of saidnucleic acid sequence is linked to a 3′ terminus of the nucleotide ornucleoside that is different from the compound of formula (VI),preferably different from the compound of formula (V) or (VI), andfurther preferably different from the compound of formula (IV); andwherein the 7′ terminus of said nucleic acid sequence is linked to a 5′terminus of the nucleotide or nucleoside that is different from thecompound of formula (VI), preferably different from the compound offormula (V) or (VI), and further preferably different from the compoundof formula (IV).

In a further preferred embodiment of the inventive oligomer, saidcompound of formula (IV) is selected from

In a further preferred embodiment, the oligomer of the invention isdouble-stranded. In a certain embodiment, exactly one or both strands ofsaid double stranded oligomer comprise at least one compound of formulae(IV), (V) or (VI).

In a third aspect, the present invention provides the inventive compoundof formula (I), (II) or (III) or the oligomer of the invention for useas a medicament in the prevention, treatment or diagnosis of a disease.

In a certain embodiment, the compound of formula (I), (II) or (III) isused as a medicament in the prevention or treatment of a disease. Theinvention provides a method of preventing a disease in a patient ortreating a patient suffering from a disease by administering atherapeutically effective amount of formula (I), (II) or (III) to thepatient. In another embodiment, the compound of formula (I), (II) or(III) is used for the manufacture of a medicament for the prevention ortreatment of a disease.

In a further embodiment, the oligomer of the invention is used as amedicament in the prevention or treatment of a disease. The inventionprovides a method of preventing a disease in a patient or treating apatient suffering from a disease by administering a therapeuticallyeffective amount of the oligomer of the invention to the patient. Inanother preferred embodiment, the oligomer of the invention is used forthe manufacture of a medicament for the prevention or treatment of adisease.

The term “patient” as used herein refers to a human or an animal,wherein the animal is preferably a mammal. The term “patient” is notrestricted to subjects showing symptoms of a disease or disorder, butincludes healthy subjects (i.e. without symptoms) or subjects being atrisk for exhibiting a symptom. A “therapeutically effective amount”refers to an amount administered to a subject, either as a single doseor as part of a series of doses, which is effective to produce a desiredphysiological response or therapeutic effect in the subject. Examples ofdesired therapeutic effects include, without limitation, improvements inthe symptoms or pathology, reducing the progression of symptoms orpathology, and slowing the onset of symptoms or pathology of thedisease. The therapeutically effective amount will vary depending on thenature of the formulation used and the type and condition of therecipient. The determination of appropriate amounts for any givencomposition is within the skill in the art, through standard series oftests designed to assess appropriate therapeutic levels. Typical andpreferred therapeutically effective amounts of the antisenseoligonucleotide range from about 0.05 to 1000 mg/kg body weight, and inparticular from about 5 to 500 mg/kg body weight.

In a further embodiment, the oligomer of the invention is an antisenseoligonucleotide. In preferred embodiment the antisense oligonucleotideof the invention is used in the prevention, treatment or diagnosis of adisease. As used herein, the term “antisense oligonucleotide” refers toan oligonucleotide that is capable of hybridizing with a target nucleicacid sequence. In a preferred embodiment, the antisense oligonucleotideis complementary to the target nucleic acid sequence. An oligonucleotideis complementary to a target nucleic acid when a sufficient number ofcomplementary positions in the oligonucleotide and the target nucleicacid are occupied by complementary nucleobases which can form hydrogenbonds with each other such that specific binding occurs between theoligonucleotide and the target nucleic acid. For example, adenine andthymine are complementary nucleobases which pair through the formationof hydrogen bonds. Hybridization can occur under varying circumstances.It is understood in the art that the sequence of an antisenseoligonucleotide need not to include nucleotides that are 100%complementary to the nucleotides of the target nucleic acid to behybridizable. An antisense oligonucleotide may hybridize over one ormore nucleotides whereas intervening or adjacent nucleotides are notinvolved in hybridization. It is preferred that the oligonucleotideportion of the antisense oligonucleotide of the present inventioncomprise at least 70% sequence complementarity to a target region withinthe target nucleic acid, more preferably that they comprise 85% or 90%sequence complementarity, and even more preferably comprise 95% sequencecomplementarity to the target region within the target nucleic acidsequence.

In a certain embodiment, the oligomer of the invention is used in theprevention or treatment of a disease, wherein the oligomer is capable ofinterfering with replication, translation, transcription, translocation,catalytic activity, complex formation, splicing or integrity of a targetnucleic acid. In a certain embodiment, the oligomer of the invention isused in the prevention or treatment of a disease, wherein the oligomeris capable of binding to a target nucleic acid, downregulatingexpression of a target nucleic acid, sterically blocking a targetnucleic acid sequence or inducing nucleic acid interference, genesilencing, degradation or exon skipping in a target nucleic acid. In apreferred embodiment, the oligomer of the invention is used in theprevention or treatment of a disease, wherein the oligomer is capable ofbinding to a target nucleic acid and downregulating expression of saidtarget nucleic acid. In another preferred embodiment, the oligomer ofthe invention is used in the prevention or treatment of a disease,wherein the oligomer is capable of binding to a target nucleic acid,sterically blocking said target nucleic acid and inducing exon skippingin said target nucleic acid. In a preferred embodiment, said targetnucleic acid is a DNA or RNA. The RNA is preferably a pre-mRNA(pre-processed or precursor messenger RNA) or mature RNA. The RNA can bean mRNA or a functional form of a non-coding RNA, such as a longnon-coding RNA, micro RNA, small interfering RNA, small nucleolar RNA,Piwi-interacting RNA, tRNA-derived small RNA, small rDNA-derived RNA,rRNA or tRNA. In a certain embodiment, the oligomer of the invention isused in the prevention or treatment of a disease, wherein the oligomeris capable of altering a splice process in a target nucleic acid,wherein preferably the target nucleic acid is a pre-mRNA. Preferablysaid oligomer is capable of inducing exon skipping in a target pre-mRNA.An “exon” refers to a defined section of a nucleic acid that encodes fora protein, or a nucleic acid sequence that is represented in the matureform of an RNA molecule after either portions of a pre-mRNA have beenremoved by splicing.

In a further embodiment, the oligomer of the invention is used as amedicament in the prevention or treatment of a disease, wherein saiddisease is a genetic disease. In a preferred embodiment, the oligomer ofthe invention is used as a medicament in the prevention or treatment ofa disease, wherein said disease is a muscular dystrophy, preferablyDuchenne muscular dystrophy. In another preferred embodiment, theoligomer for use as a medicament in the prevention, treatment ordiagnosis of a disease is the nucleic acid sequence of SEQ ID NO: 21 orthe nucleic acid sequence of SEQ ID NO: 24, preferably the nucleic acidsequence of SEQ ID NO: 24 and wherein the disease is a musculardystrophy, preferably Duchenne muscular dystrophy. The oligomers of theinvention and in particular SEQ ID NO: 24 maintain a good affinitytoward RNA, and oligomers consisting of compounds of formula (V) seem toconfer a significantly improved biostability compared to its naturallyoccurring corresponding DNA. Moreover, the nucleic acid of SEQ ID NO: 24does not activate complement significantly, and complement activationrepresents an important toxic response often associated with an in vivouse of antisense oligonucleotides. Therefore, the oligomers of theinvention, preferably oligomers comprising or preferably consisting ofcompounds of formula (V) are promising antisense candidates. Finally,oligomers consisting of compounds of formula (V), preferably the nucleicacid of SEQ ID NO: 24 is able to induce strong exon skipping of exon 23and double exon skipping of exons 22 and 23. These promising resultsindicate that the oligomer of the invention, especially oligomersconsisting of compounds of formula (V) meet the prerequisites to inducea strong therapeutic effect in patients suffering muscular dystrophies,such as Duchenne.

In another aspect, the present invention provides for the oligomer ofthe invention for use as a medicament in the prevention or treatment ofa disease.

In a further aspect, the oligomer of the invention is used in thediagnosis of a disease. In a further aspect, the oligomer of theinvention is used as a medicament in the diagnosis of a disease. Inanother preferred embodiment, the oligomer of the invention is used forthe manufacture of a medicament for the diagnosis of a disease. Theinvention provides a method of diagnosing a disease in a patient. Saiddiagnosis or said diagnosing comprises

(i) administering an effective amount of the oligomer of the inventionto a patient, wherein the oligomer is labelled, and

(ii) non-invasive or invasive, preferably non-invasive in vivo imagingof the labelled oligomer or

(i′) obtaining a sample from a patient,

(ii′) adding a oligomer of the invention to the sample, wherein theoligomer is labelled, and

(iii′) analyzing the sample for binding of the labelled oligomer withnucleic acids included in the sample. In a preferred embodiment, theoligomer of the invention used in the diagnosis of a disease is anoligonucleotide, more preferably an antisense oligonucleotide. Thesample obtained from a patient is preferably a blood, serum, liquor ortissue sample. The term “labelled oligomer” as used herein refers to anoligomer comprising a label. Preferably the label is selected from afluorescent label, dye, reporter group or a radiolabel.

In a further aspect, the invention provides a pharmaceutical compositioncomprising at least one compound selected from formula (I), (II) or(III). In a further aspect, the invention provides a pharmaceuticalcomposition comprising at least one oligomer of the invention. In apreferred embodiment, said pharmaceutical composition comprises one ormore oligomers of the invention, wherein at least one of said one ormore oligomers is an oligonucleotide, more preferably an antisenseoligonucleotide. In a preferred embodiment, the pharmaceuticalcomposition comprises a therapeutically-effective amount of the at leastone compound of formula (I), (II) or (III) or the at least one oligomerof the invention, preferably formulated together with one or morepharmaceutically acceptable carriers. In one embodiment, a unit dose ofthe pharmaceutical composition of the invention contains about 1microgram to 20,000 micrograms of the oligomer or the compound offormula (I), (II) or (III) per unit, and preferably from about 10 to1000 micrograms. For intravenous delivery, a unit dose of thepharmaceutical formulation contains preferably from 0.5 to 500micrograms per kg body weight, more preferably from 5 to 300 microgramsper kg body weight of the oligomer of the invention. In thepharmaceutical compositions of the invention, the oligomer or thecompound of formula (I), (II) or (III) is ordinarily present in anamount of about 0.5-95% by weight based on the total weight of thecomposition.

In a preferred embodiment, the pharmaceutical composition comprising theat least one compound of formula (I), (II) or (III) or the at least oneoligomer of the invention further comprises at least onepharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” as used herein means a pharmaceutically acceptablematerial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium,calcium or zinc stearate, or steric acid), or solvent encapsulatingmaterial. The pharmaceutically acceptable carrier can be involved incarrying or transporting the subject compound from one organ or portionof the body to another. Methods for the delivery of nucleic acids aredescribed, for example, in Akhtar et al., 1992, Trends Cell Bio., 2:139;and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.Akhtar; Sullivan et al., PCT WO 94/02595. These and other protocols canbe utilized for the delivery of virtually any nucleotide or nucleic acidmolecule, including the compound of formula (I), (II) or (III) and theoligomers of the present invention. The invention also features thepharmaceutical composition of the invention further comprisingP-glycoprotein inhibitors (such as Pluronic P85), which can enhanceentry of drugs into various tissues; biodegradable polymers, such aspoly (DL-lactide-coglycolide) microspheres for sustained releasedelivery after implantation (Emerich, D F et al., 1999, Cell Transplant,8, 47-58) Alkermes, Inc. Cambridge, Mass.); nanoparticles, such as thosemade of polybutylcyanoacrylate, which can deliver drugs across the bloodbrain barrier and can alter cellular uptake mechanisms (FrogNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999); or liposomescontaining polyethylene glycol-lipids.

Administration of the pharmaceutical composition, the compound offormula (I), (II) or (III) or the oligomer of the invention can becarried out using the various mechanisms known in the art. In apreferred embodiment, the pharmaceutical composition, the compound offormula (I), (II) or (III) or the oligomer of the invention isadministered locally or systemically. In a preferred embodiment, thepharmaceutical composition, the compound of formula (I), (II) or (III)or the oligomer of the invention is administered orally (for example, asan aqueous or non-aqueous solution or suspension, tablet, bolus, powder,granule, paste), parenterally (for example, by subcutaneous,intramuscular, intravenous, intraperitoneal or epidural injection as,for example, a sterile solution or suspension, or sustained-releaseformulation), topically (for example, as a cream, ointment, or acontrolled-release patch or spray), intravaginally or intrarectally (forexample, as a pessary, cream or foam), sublingually, ocularly,transdermally, nasally, intracellularly or by direct local tumorinjection. In a preferred embodiment, the pharmaceutical compositioncomprising the oligomer of the invention is used for downregulatingexpression of a target nucleic acid, sterically blocking a targetnucleic acid sequence or inducing nucleic acid interference, genesilencing, degradation or exon skipping in a target nucleic acid.

In a further aspect, the oligomer of the invention is used in vitro forbinding to a target nucleic acid sequence. In a preferred embodiment,the oligomer of the invention is used in vitro for downregulatingexpression of a target nucleic acid, sterically blocking a targetnucleic acid sequence or inducing nucleic acid interference, genesilencing, degradation or exon skipping in a target nucleic acid. In acertain embodiment, the oligomer of the invention is used in vitro forinterfering with replication, translation, transcription, translocation,catalytic activity, complex formation, splicing or integrity of a targetnucleic acid. In a preferred embodiment, oligomer of the invention isused in vitro for binding to a target nucleic acid, wherein exonskipping is induced in said target nucleic acid. In a preferredembodiment, the present invention provides an in vitro method ofdown-regulating the expression of a target gene in the cytosol of a cellby delivering the oligomer of the invention of the invention across amembrane of the cell. In a preferred embodiment, the target nucleic acidis DNA, pre-mRNA or mature mRNA.

In a further aspect, the invention provides a method for solid-phasesynthesis of an oligomer of the invention comprising the use of any oneof the compounds of formulae (I) to (VI).

EXAMPLES

General Procedures

All reactions were performed in dried glassware and under an inertatmosphere of Argon. Anhydrous solvents for reactions were obtained byfiltration through activated alumina or by storage over molecular sieves(4 Å). Colum chromatography (CC) was performed on silica gel (SiliaFlashP60, 40-63 um, 60 Å). Methanol used for CC was of HPLC grade, all othersolvents used for CC were of technical grade and distilled prior to use.Thin-layer chromatography was performed on silica gel plates(macherey-nagel, pre-coated TLC-plates sil G-25 UV254). Compounds werevisualized under UV-light or by dipping in a p-Anisaldehyde stainingsolution [p-Anisaldehyde (3.7 mL), glacial acetic acid (3.7 mL),concentrated sulfuric acid (5 mL), ethanol (135 mL)] followed by heatingwith a heat gun. NMR spectra were recorded at 300 or 400 MHz (¹H), at 75or 101 MHz (¹³C) and at 122 MHz (³¹P) in either CDCl₃, CD₃OD or CD₃CN.Chemical shifts (δ) are reported relative to the residual undertreatedsolvent peak [CDCl₃: 7.26 ppm (¹H), 77.16 ppm (¹³C); CD₃OD: 3.31 ppm(¹H), 49.00 ppm (¹³C)]. Signal assignments are based APT and DEPT and on¹H, ¹H and ¹H, ¹³C correlation experiments (COSY, HSQC, HMBC). Highresolution mass detections were performed by electrospray ionization inthe positive mode (ion trap, ESI⁺).

Within this Examples section, for sake of simplicity, nucleotides ornucleosides mentioned in this Examples section refer to beta anomers,unless mentioned specifically as alpha anomers. Furthermore, andconsistent hereto, oligomers or oligonucleotides mentioned within thisExamples section comprise beta anomers, unless mentioned specifically asalpha anomers.

Temperature of Melting

UV-melting experiments were recorded on a Varian Cary Bio 100 UV/visspectrophotometer. Experiments were performed at 2 μM duplexconcentration, 10 mM NaH₂PO₄, between 0 M and 150 mM NaCl (alpha anomer)or between 0.05 M and 1.00 M NaCl (beta anomer) and pH adjusted to 7.0.Samples were protected from evaporation by a covering layer ofdimethylpolysiloxane. Absorbance was monitored at 260 nm. For everyexperiment, three cooling-heating cycles were performed with atemperature gradient of 0.5° C./min. The maxima of the curves firstderivative were extracted with Varian WinUV software and T_(m) valueswere reported as the average of the six ramps.

Circular Dichroism Spectroscopy

CD-spectra were recorded on a Jasco J-715 spectropolarimeter equippedwith a Jasco PFO-3505 temperature controller. Sample conditions were thesame as for UV-melting experiments. Spectra were recorded between 210and 320 nm at a 50 nm/min rate and the temperature was measured directlyfrom the sample. For each experiment, a blank containing the same saltconcentrations as the sample were recorded. The reported spectra wereobtained by taking a smoothed average of three scans and subtracting thecorresponding blank spectrum.

Characterizations of Oligonucleotides

SEQ ID Exact Experimental Entry NO Sequence^(a) mass mass ON1  1d(GGATGTTCtCGA) 3700.67 3701.66 ON2  2 d(GGAtGTTCTCGA) 3700.67 3701.66ON3  3 d(GGATGttCTCGA) 3726.68 3727.70 ON4  4 d(GGATGTT c TCGA) 3714.683715.67 ON5  5 d(GGATGTTCT c GA) 3714.68 3715.67 ON6  6 d(GGaTGTTCTCGA)3700.67 3701.66 ON7  7 d(GGATgTTCTCGA) 3700.67 3701.66 ON8  8d(GGATGTTcTCGA) 3700.67 3701.66 ON9  9 d(GGATGTTCTcGA) 3700.67 3701.66ON10 10 d(GGATGTTcTcGA) 3726.68 3727.67 ON11 11 d(GCAttt ttACCG) 3739.713740.72 ON12 12 5′-(ttt t c t  cc t)-7′ 2905.65 2906.64 ON13 135′-(gga tgt t c t  c ga)-7 4014.87 4015.85 ON14 14 5′-(t c g aga a c a tcc )-7′ 3980.91 3981.90 ON15 15 5′-( cc t a c a aga g c t)-7 3980.913981.90 ON16 16 5′-d(GGA TGT TCt CGA)-3′ 3700.67 3701.64 ON17 175′-d(GGA t GT TCT CGA)-3′ 3700.67 3701.64 ON18 185′-d(GGA tGT TCt CGA)-3′ 3726.68 3727.66 ON19 195′-d(GGA TGt tCT CGA)-3′ 3726.68 3727.66 ON20 205′-d(GCA ttt ttA CCG)-3′ 3739.71 3739.65 ON21 215′-d(agc tct tgt agg)-7′ 4014.87 4015.86 ON22 225′-d(cct aca aga gct)-7′ 3980.91 3981.90 ON23 235′-d(tcg aga aca tcc)-7′ 3980.91 3981.90 ON24 245′-d(t*c*c*a*t*t*c*g*g*c*t*c*c*a*a*)-7′ 5185.81 5186.81 ^(a)A, G, T, Cdenote natural 2′-deoxynucleosides; a, g,t, c corresponds to modifiedadenine, guanine, thymine and methylcytosine respectively, *denotes aphosphorothioate linkage, c stands for the modified 5-methyl cytosinenucleoside.

Example 1 Syntheses of the Inventive Compounds General Overview

The bicyclic scaffolds 7 and 10 envisaged for subsequent nucleosidesynthesis could be constructed from the previously describedintermediate 1 (Tarköy, M.; Bolli, M.; Schweizer, B.; Leumann, C. Helv.Chim. Acta 1993, 76, 481) (Scheme 1). The epoxide ring in 1 wasefficiently opened by LiHMDS mediated intramolecular elimination at −78°C., yielding the unsaturated ester 2 in good yield. Subsequentnickel-catalyzed NaBH₄ reduction of 2 proceeded stereospecifically fromthe convex side of the bicyclic core structure, resulting in ester 3 asthe only identifiable diastereoisomer. The hydroxyl function in 3 wasthen TBDPS protected, giving 4 in quantitative yield. Intermediate 4 wasconsequently reduced with DIBAL at −78° C., leading to aldehyde 5. Theacetonide protecting group in 5 was then hydrolyzed under mildconditions with In(OTf)₃ as catalyst (Golden, K. C.; Gregg, B. T.;Quinn, J. F. Tetrahedron Lett. 2010, 51, 4010), in a mixture of MeCN andH₂O, and the resulting bicyclic hemiacetal converted into the methylglycoside 6 by simply changing the solvent to MeOH. Compound 6 was thenacetylated to afford the protected precursor 7 that was used for thesynthesis of the corresponding purine nucleosides via Vorbrüggenchemistry.

The synthesis of the preferred pyrimidine nucleosides of the presentinvention consisted in the well-established application of theβ-stereoselective NIH induced addition of the nucleobases to acorresponding bicyclic glycal (Medvecky, M.; Istrate, A.; Leumann, C. J.J. Org. Chem. 2015, 80, 3556; Dugovic, B.; Leumann, C. J. Journal ofOrganic Chemistry 2014, 79, 1271; Lietard, J.; Leumann, C. J. J. Org.Chem. 2012, 77, 4566). First, to introduce the thymine nucleobase, theN-iodosuccinimide (NIS) induced nucleosidation was performed on thedirect precursor of glycal 8, where R₁=TMS, that was easily obtainedfrom 6 by treatment with TMSOTf only. This approach resulted in thestereoselective formation of the corresponding β-nucleoside, however,with a significant contamination of 7% of the α-anomer that remainedinseparable by standard chromatography techniques. It was reasoned thatthe β-selectivity could be enhanced by increasing steric bulk at R₁ anddecreasing it at R₂, as in glycal 10. This would favor initial α-attackof the electrophilic iodine at C(4). To this end compound 6 wasconverted to glycal 8 with TMSOTf followed by a short treatment withTBAF to remove the newly introduced TMS group selectively. Intermediate8 was then elaborated into the dimethoxytrityl compound 9 which wasfinally subjected to removal of the TBDPS protecting group with TBAF togive the desired sugar component 10.

NIS-nucleosidation on the in situ TMS protected glycal 10, followed byradical reduction of the iodide intermediate with Bu₃SnH, yielded theDMTr-protected thymidine derivative 11 in good yield containing onlytrace amounts (<2% by ¹H-NMR) of the α-anomer (Scheme 2). Finalphosphitylation with 2-cyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite lead to the thymidinephosphoramidite building block 12. The synthesis of the 5-methylcytosinenucleoside was achieved by conversion of the base thymine. To this end,nucleoside 11 was TMS protected and converted to the correspondingtriazolide by treatment with 1,2,4-triazole and POCl₃. Subsequenttreatment of this triazolide in a mixture of ammonia and 1,4-dioxaneyielded the corresponding 5-methylcytosine nucleoside, which wasdirectly protected with Bz₂O to give 13 in 88% yield over three steps.The phosphoramidite 14 was obtained by a phosphitylation as describedabove.

Classical Vorbrüggen nucleosidation was applied for introducing thepurine nucleobases resulted generally in the prevalence of theα-nucleosides. The conversion of precursor 7 with eitherN⁶-benzoyladenine or 2-amino-6-chloropurine leads to the inseparableanomeric mixtures 15 and 20, resp. in α/β ratios of 4:1 and 7:3 (Scheme3). Separation of anomers was possible after deacetylation, leading tothe pure β-anomers 16 and 21. From here, the adenine building block 19could be obtained by standard dimethoxytritylation (→17) followed TBAFmediated cleavage of the silyl protecting group (→19) andphosphitylation. The synthesis of the guanine building block requiredthe conversion of the 2-amino-6-chloropurine nucleobase. This wasachieved by treatment of 21 with 3-hydroxypropionitrile and TBD andsubsequent protection of the 2-amino group with DMF, yielding theprotected guanosine derivatives 22. Following the same chemical pathwayas above, the synthesis of the guanine building block 25 was achieved bydimethoxytritylation (→23) followed by removal of silyl protecting group(→24) and phosphitylation.

Starting from protected sugar 7 the synthesis of four preferredphosphoramidite building blocks of the present invention was developed.Treatment of a mixture of sugar 7 and in situ silylated thymine withTMSOTf resulted in the smooth formation of the nucleoside 35, with afavorable anomeric ratio α/β of approximately 85:15 (determined by¹H-NMR) (Scheme 4). The chemical pathway leading to the thymidinephosphoramidite bearing the DMTr group on the 5′ position does not allowthe separation of anomers by standard chromatography. Therefore, and inorder to introduce the modification with polarity reversal into DNAstrands, the DMTr group was introduced on the 7′ position. To this end,the silyl group of 35 was removed by short treatment with TBAF (→36)followed by standard dimethoxytritylation (→37). Separation of the twoanomers was possible after standard deacetylation, leading to the pureα-anomer 38. The thymidine building block 39 was finally obtained byphosphitylation with 2-cyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite in the presence of5-(Ethylthio)-1H-tetrazole. The intermediate 38 also offered us shortaccess to the 5-methylcytosine nucleoside, by conversion of the in situTMS protected nucleoside 38 to the corresponding triazolide with POCl₃and 1,2,4-triazole, followed by treatment in a mixture of ammonia and1,4-dioxane. Direct protection with Bz₂O in DMF resulted in theefficient formation of nucleoside 40, the labile silyl protecting groupbeing cleaved during the process. Final phosphitylation in conditions asdescribed above afforded the 5-methylcytidine phosphoramidite 41.

For the purine nucleobases, the introduction of the purines wereperformed by a short nucleosidation in slightly elevated temperaturewith either N⁶-benzoyladenine or 2-amino-6-chloropurine, leading to thenucleoside 15 and 20, resp. in α/β ratios of 4:1 and 7:3 (Scheme 5). Toseparate the anomers, acetyl groups were removed under mild conditions,yielding the pure α-anomers 42 and 48. The formation of the adenosinebuilding block continues with the reintroduction of the acetylprotecting group (→43), removal of the TPDPS protecting group with TBAF(→44) followed by standard dimethoxytritylation (→45). Selectivedeprotection of the acetyl group (→46) followed by phosphitylation inconditions as described above yielded the adenine building block 47.

For the guanine building block, after separation of the two anomers, the6-chloropurine was converted to the guanine nucleobase by treatment withTBD and 3-hydroxypropionitrile yielding the guanosine nucleoside 49.Acetylation over 48 h allowed the concomitant protection of the5′-hydroxy and 2-amino groups, yielding the protected nucleoside 50.Similarly as above, the DMTr group was introduced by removal of thesilyl protecting group with TBAF (→51) followed by dimethoxytritylation(→52). The two acetyl groups were removed by treatment with K₂CO₃ andthe resulting polar product was directly protected with DMF to affordthe guanosine nucleoside 53. Final phosphitylation yielded buildingblock 54.

Example 2 Ethyl (E and Z,1′R,5′S,7′R)-(7′-hydroxy-3′,3′-dimethyl-2,4′-dioxabicyclo[3.3.0]oct-6′-ylidene)acetate(2a/b)

A solution of the epoxide 1 (4.46 g, 18.4 mmol) in dry THF (100 mL) wascooled down to −78° C. Then LiHMDS (1M in THF, 22.1 mL, 22.1 mmol) wasslowly added. The solution was stirred for 2 hours at −78° C. beforebeing allowed to warm to rt and neutralized with addition of 1M aqueousHCl (22.1 mL). The mixture was then diluted with EtOAc (100 mL) and THFwas removed under reduced pressure. The mixture is then washed with 0.5M NaH₂PO₄ (50 mL) and aqueous phase extracted with EtOAc (2×50 mL). Thecombined organic phases were dried over MgSO₄, filtered and evaporated.The crude product was purified by CC (EtOAc/hexane 3:1) to yield the twoisomers 2a/b (3.30 g, 74%) as a slightly yellow solid.

Data for 2a: R_(f)=0.37 (EtoAc/hexane 1:1);

¹H NMR (300 MHz, CDCl₃) δ 6.07-5.98 (m, 1H, H—C(2)), 5.59 (d, J=6.0 Hz,1H, H—C(5′)), 4.94-4.81 (m, 1H, H—C(1′)), 4.65 (t, J=5.6 Hz, 1H,H—C(7′)), 4.18 (q, J=7.1 Hz, 2H, CH₃CH₂), 2.67 (br, 1H, OH), 2.37 (dd,J=13.5, 7.5 Hz, 1H, H—C(8′)), 1.55-1.42 (m, 1H, H—C(8′)), 1.40, 1.33(2s, 6H, (CH₃)₂C), 1.26 (t, J=7.1 Hz, 3H, CH₂CH₃).

¹³C NMR (75 MHz, CDCl₃) δ 165.75 (C(1)), 161.61 (C(6′)), 116.53 (C(2)),110.69 (C(3′)), 76.55 (C(5′)), 75.52 (C(1′)), 71.63 (C(7′)), 60.51(CH₂CH₃), 37.46 (C(8′)), 26.44, 24.11 ((CH₃)₂C), 14.27 (CH₂CH₃).

ESI⁺-HRMS m/z calcd for C₁₂H₁₉O₅ ([M+H]⁺) 243.1227, found 243.1231.

Data for 2b: R_(f)=0.52 (EtoAc/hexane 1:1);

¹H NMR (300 MHz, CDCl₃) δ 6.15-6.05 (m, 1H, H—C(2)), 5.37-5.02 (m, 2H,H—C(5′), OH), 4.87 (d, J=3.4 Hz, 1H, H—C(1′)), 4.67 (t, J=4.9 Hz, 1H,H—C(7′)), 4.20 (qd, J=7.1, 0.9 Hz, 2H, CH₃CH₂), 2.55 (dd, J=14.6, 8.1Hz, 1H, H—C(8′)), 1.94-1.77 (m, 1H, H—C(8′)), 1.39-1.25 (m, 9H, (CH₃)₂C,CH₂CH₃).

¹³C NMR (75 MHz, CDCl₃) δ 167.91 (C(1)), 167.43 (C(6′)), 120.13 (C(2)),111.75 (C(3′)), 81.62 (C(5′)), 78.08 (C(1′)), 70.85 (C(7′)), 61.25(CH₂CH₃), 36.53 (C(8′)), 27.38, 25.45 ((CH₃)₂C), 14.19 (CH₂CH₃).

ESI⁺-HRMS m/z calcd for C₁₂H₁₉O₅ ([M+H]⁺) 243.1227, found 243.1227.

Example 3 Ethyl(1′R,5′S,6'S,7′R)-(7′-hydroxy-3′,3′-dimethyl-2,4′-dioxabicyclo[3.3.0]oct-6′-yl)acetate(3)

To a solution of the alcohols 2a/b (12.65 g, 52.2 mmol) and nickelchloride hexahydrate (2.48 g, 10.4 mmol) in EtOH (300 mL) was addedportion wise sodium borohydride (9.88 g, 261 mmol) at 0° C. Theresulting dark solution was stirred for 30 min at 0° C. and 90 min atrt. Then EtOH was carefully concentrated under reduced pressure, theresulting solid diluted with EtOAc (200 mL) and the excess of NaBH₄quenched by addition of water (100 mL) at 0° C. followed by stirring atrt for 30 min. The two phases are then separated. Organic phase waswashed with water (100 mL). Aqueous phases are then combined, filteredand extracted with EtOAc (2×100 mL). The combined organic phases weredried over MgSO₄, filtered and concentrated. The crude product waspurified by CC (EtOAc/hexane 2:1) to yield 3 (11.4 g, 90%) as a whitesolid.

Data for 3: R_(f)=0.40 (EtOAc/hexane 1:1);

¹H NMR (300 MHz, CDCl₃) δ 4.65-4.52 (m, 2H, H—C(1′), H—C(5′)), 4.15 (qd,J=7.1, 1.4 Hz, 2H, CH₃CH₂), 4.05 (ddd, J=10.0, 9.99, 6.2 Hz, 1H,H—C(7′)), 2.86 (br, s, 1H, OH), 2.65 (qd, J=16.9, 7.1 Hz, 2H, H—C(2)),2.24 (dd, J=13.7, 6.2 Hz, 1H, H—C(8′)), 1.93 (dt, J=12.7, 7.1 Hz, 1H.H—C(6′)), 1.56 (ddd, J=13.9, 10.2, 5.5 Hz, 1H, H—C(8′)), 1.38 (s, 3H,(CH₃)₂C), 1.30-1.21 (m, 6H, (CH₃)₂C, CH₂CH₃).

¹³C NMR (75 MHz, CDCl₃) δ 174.38 (C(1)), 109.06 (C(3′)), 79.65 (C(5′)),77.19 (C(1′), 74.32 (C(7′), 60.80 (CH₂CH₃), 46.66 (C(6′)), 40.38(C(8′)), 32.43 (C(2)), 26.00, 23.69 ((CH₃)₂C), 14.17 (CH₂CH₃).

ESI⁺-HRMS m/z calcd for C₁₂H₂₁O₅ ([M+H]⁺) 245.1384, found 245.1388.

Example 4 Ethyl(1′R,5′S,6′S,7′R)-(7′-(tert-butyldiphenylsilyl)oxy)-3′,3′-dimethyl-2,4′-dioxabicyclo[3.3.0]oct-6′-yl)acetate(4)

To a solution of the alcohol 3 (2.50 g, 10.2 mmol), N-methylimidazole(12.6 g, 153 mmol) and iodine (7.80 g, 30.6 mmol) in dry THF (60 mL) wasadded dropwise tert-butyl(chloro)diphenylsilane (3.0 mL, 11.2 mmol) atrt. The solution was stirred for 3 hours at rt and hen THF wasevaporated, the mixture diluted with EtOAc (50 mL) and washed with 10%aqueous Na₂O₃S₂ (2×40 mL). Aqueous phases are then combined andextracted with EtOAc (50 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (EtOAc/hexane 1:10) to yield 4 (5.01 g, quantitative yield) as awhite solid

Data for 4: R_(f)=0.87 (DCM/MeOH 10:1);

¹H NMR (300 MHz, CDCl₃) δ 7.77-7.59 (m, 4H, H-arom), 7.51-7.32 (m, 6H,H-arom), 4.61 (t, J=5.7 Hz, 1H, H—C(5′)), 4.49 (t, J=5.7 Hz, 1H,H—C(1′)), 4.15 (q, J=6.9 Hz, 2H, CH₃CH₂), 3.96 (dd, J=15.5, 9.5 Hz, 1H,H—C(7′)), 2.64-2.32 (m, 2H, H—C(2)), 2.15 (tt, J=9.0, 4.3 Hz, 1H,H—C(6′)), 1.83 (dd, J=12.7, 5.2 Hz, 1H, H—C(8′)), 1.61-1.45 (m, 1H,H—C(8′)), 1.27 (td, J=7.1, 1.9 Hz, 3H, CH₂CH₃), 1.18 (s, 6H, (CH₃)₂C),1.09, 1.08 (2s, 9H, (CH₃)₃—C—Si)

¹³C NMR (75 MHz, CDCl₃) δ 173.07 (C(1)), 135.87, 135.85 (CH-arom),134.08, 133.73 (C-arom), 129.80, 129.75, 127.67, 127.58 (CH-arom),108.82 (C(3′)), 77.92 (C(5′)), 76.96 (C(1′)), 74.93 (C(7′)), 60.24(CH₂CH₃), 47.27 (C(6′)), 40.27 (C(8′)), 31.10 (C(2)), 27.04(CH₃)₃—C—Si), 25.86 ((CH₃)₂C), 23.83 ((CH₃)₂C), 19.23 (CH₃)₃—C—Si),14.24 (CH₂—CH₃).

ESI⁺-HRMS m/z calcd for C₂₈H₃₉O₅Si ([M+H]⁺) 483.2561, found 483.2562.

Example 5(1′R,5′S,6′S,7′R)-(7′-(tert-butyldiphenylsilyl)oxy)-3′,3′-dimethyl-2,4′-dioxabicyclo[3.3.0]oct-6′-yl)acetaldehyde(5)

A solution of the ester 4 (8.56 g, 16.3 mmol) in dry DCM (120 mL) wascool down to −78° C. and then DiBAL-H (1M in cyclohexane, 18 mL, 18mmol) was slowly added. The solution was further stirred at −78° C. for90 min before being allowed to warm to rt. Reaction was quenched byaddition of 0.5 M aqueous NaH₂PO₄ (100 mL). The organic phase wasseparated and aqueous phase was further extracted with DCM (2×100 mL).The combined organic phases were dried over MgSO₄, filtered andevaporated. The crude product was purified by CC (EtOAc/hexane 2:10 to2:1) to yield aldehyde 5 (6.36 g, 89%) and alcohol 34 (0.637 g, 9%).

Data for 5: R_(f)=0.65 (EtOAc/hexane 2:1);

¹H NMR (300 MHz, CDCl₃) δ 9.72 (s, 1H, H—C(1)), 7.65 (td, J=8.0, 1.6 Hz,4H, H-arom), 7.47-7.33 (m, 6H, H-arom), 4.57 (t, J=5.7 Hz, 1H, H—C(5′)),4.51 (t, J=5.7 Hz, 1H, H—C(1′)), 3.99 (td, J=10.0, 5.9 Hz, 1H, H—C(7′)),2.58-2.43 (m, 2H, H—C(2)), 2.20-2.08 (m, 1H, H—C(6′)), 1.87 (dd, J=13.5,5.9 Hz, 1H, H—C(8′)), 1.53 (ddd, J=13.5, 10.1, 5.5 Hz, 1H, H—C(8′)),1.16 (d, J=3.5 Hz, 6H, ((CH₃)₂C), 1.05 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 201.87 (C(1)), 135.93, 135.90 (CH-arom),133.96, 133.73 (C-arom), 129.96, 129.89, 127.79, 127.68 (CH-arom),108.89 (C(3′)), 77.76 (C(5′)), 77.17 (C(1′)), 74.96 (C(7′), 45.44(C(6′)), 41.31 (C(2)), 40.16 (C(8′)), 27.08 (CH₃)₃—C—Si), 25.87((CH₃)₂C), 23.79 ((CH₃)₂C), 19.25 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₂₆H₃₅O₄Si ([M+H]⁺) 439.2299, found 439.2297.

Example 6(3aR,4R,6R,6aS)-4-((tert-butyldiphenylsilyl)oxy)-2-methoxyhexahydro-2H-cyclopenta[b]furan-6-ol(6)

To a solution of the aldehyde 5 (13.73 g, 31.31 mmol) in MeCN (170 mL)and H₂O (19 mL) was added Indium(III) trifluoromethanesulfonate (703 mg,1.25 mmol). The solution was further stirred for 48 hours, and thensolvents were removed under reduced pressure and coevaporated withtoluene. The residue was dissolved in dry MeOH and stirred for 6 hours.After evaporation of solvent, the crude product was purified by CC(EtOAc/hexane 3:10) to yield a mixture of 6 (10.50 g, 81%) in ananomeric ratio α/β≈4:1 as a colorless oil.

Data for 6: R_(f)=0.53 (EtOAc/hexane 1:1);

¹H NMR (300 MHz, CDCl₃) δ 7.63 (dd, J=7.1, 0.6 Hz, 4H, H-arom),7.46-7.34 (m, 6H, H-arom), 4.98 (d, J=4.8 Hz, 0.8H, H—C(2)), 4.91 (dd,J=5.9, 1.3 Hz, 0.2H, H—C(2)), 4.63-4.54 (m, 1H, H—C(6a)), 4.53-4.37 (m,1H, H—C(6)), 4.09 (m, 0.2H, H—C(4)), 3.92 (br, 0.8H, H—C(4)), 3.29, 3.27(2s, 3H, MeO), 2.79 (dd, J=17.0, 8.2 Hz, 0.8H, H—C(3a)), 2.64-2.51 (m,0.2H, H—C(3a)), 2.29 (d, J=8.1 Hz, 1H, OH), 2.10-1.80 (m, 2.4H, H—C(3),H—C(5)), 1.65 (ddd, J=13.2, 9.1, 4.4 Hz, 0.8H, H—C(5)), 1.44-1.34 (m,0.2H, H—C(3)), 1.22 (ddd, J=13.2, 8.1, 4.9 Hz, 0.8H, H—C(3)), 1.05 (s,9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 135.78, 135.74 (CH-arom), 133.96, 133.84(C-arom), 129.78, 127.72 (CH-arom), 107.21, 106.50 (C(2)), 85.37, 81.76(C(6a)), 78.11, 77.19 (C(4)), 73.03, 72.44 (C(6)), 55.30, 54.46 (MeO),50.91, 49.67 (C(3a)), 41.13, 40.29 (C(3)), 38.16, 37.98 (C(5)), 26.96,26.92 (CH₃)₃—C—Si), 19.07 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₂₆H₃₅O₄Si ([M+H]⁺) 435.1962, found 435.1950.

Example 7(3aR,4R,6R,6aS)-4-((tert-butyldiphenylsilyl)oxy)-2-methoxyhexahydro-2H-cyclopenta[b]furan-6-ylacetate (7)

To a solution of sugar 6 (3.35 g, 8.12 mmol) and 4-Dimethylaminopyridine(1.29 g, 10.6 mmol) in dry DCM (100 mL) was added acetic anhydride (3.8mL, 41 mmol) at rt. After stirring for 2 h, reaction is quenched by slowaddition of satd NaHCO₃ (10 mL). The mixture is then diluted with satdNaHCO₃ (50 mL) and extracted with DCM (3×50 mL). The combined organicphases were dried over MgSO₄, filtered and evaporated. The crude productwas purified by CC (EtOAc/Hexanne 1:2) to yield a mixture of 7 (3.53 g,96%) in an anomeric ratio α/β≈4:1 as a colorless oil.

Data for 7: R_(f)=0.42 (EtOAc/hexane 1:2);

¹H NMR (400 MHz, CDCl₃) δ 7.70-7.59 (m, 4H, H-arom), 7.48-7.34 (m, 6H,H-arom), 5.41 (dt, J=11.0, 5.6 Hz, 0.8H, H—C(6)), 5.28 (ddd, J=11.7,6.6, 5.2 Hz, 0.2H, H—C(6)), 4.99 (d, J=4.8 Hz, 0.8H, H—C(2)), 4.89-4.81(m, 0.4H, H—C(2), H—C(6a)), 4.76-4.69 (m, 0.8H, H—C(6a)), 4.11 (d, J=5.1Hz, 0.2H, H—C(4)), 3.90 (d, J=4.0 Hz, 0.8H, H—C(4)), 3.27, 3.24 (2s, 3H,MeO), 2.81 (dd, J=16.6, 7.6 Hz, 0.8H, H—C(3a)), 2.60 (dd, J=10.1, 7.0Hz, 0.2H, H—C(3a)), 2.30-2.18 (m, 0.2H, H—C(5)), 2.12, 2.10 (2s, J=4.7Hz, 3H, MeCO₂), 2.07-1.82 (m, 2.8H, H—C(5), H—C(3)), 1.24 (ddd, J=12.9,7.6, 3.7 Hz, 1H, H—C(3)), 1.07 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 170.75, 170.66 (MeCO₂), 135.77, 135.73, 135.72(CH-arom), 133.75, 133.65 (C-arom), 129.82, 129.74, 127.76, 127.75,127.71 (CH-arom), 106.19, 106.15 (C(2)), 83.17, 79.80 (C(6a)), 77.49,76.46 (C(4)), 75.64, 74.41 (C(6)), 54.34, 54.25 (MeO), 51.48, 50.17(C(3a)), 38.05, 37.98 (C(3)), 36.96, 36.21 (C(5)), 26.95, 26.90(CH₃)₃—C—Si), 21.09, 21.04 (MeCO₂), 19.04 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₂₆H₃₄O₅NaSi ([M+Na]⁺) 477.2068, found 477.2063.

Example 8(3aR,4R,6R,6aS)-4-((tert-butyldiphenylsilyl)oxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-6-ol(8)

To a solution of the sugar 6 (2.08 g, 5.04 mmol) in dry DCM (35 mL) wasadded 2,6-lutidine (2.95 mL, 25.2 mmol) at 0° C. After stirring for 20min at 0° C., TMSOTf (2.73 mL, 15.1 mmol) was added dropwise and thenthe solution was allowed to warm to rt and stirred for another 60 min.The reaction was then quenched by addition of satd NaHCO₃ (40 mL). Theorganic phase was separated and aqueous phase was further extracted withDCM (3×30 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated.

The resulting product was dissolved in dry THF (35 mL), cool down to 0°C., and TBAF (1M in THF, 5.6 mL, 5.6 mmol) was added. The solution wasstirred for 10 min and then diluted with satd NaHCO₃ (30 mL) andextracted with DCM (4×40 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (EtOAc/hexane 1:4) to yield the glycal 8 (1.76 g, 92%).

Data for 8: R_(f)=0.49 (EtOAc/hexane 1:2);

¹H NMR (300 MHz, CDCl₃) δ 7.66 (m, 4H, H-arom), 7.42 (m, 6H, H-arom),6.22 (t, J=2.1 Hz, 1H, H—C(2)), 4.91 (dd, J=8.2, 5.3 Hz, 1H, H—C(3)),4.70 (dt, J=11.1, 5.6 Hz, 1H, H—C(6)), 4.56 (t, J=2.8 Hz, 1H, H—C(6a)),3.97 (d, J=4.0 Hz, 1H, H—C(4)), 3.24 (d, J=8.2 Hz, 1H, H—C(3a)), 2.30(br, 1H, OH), 2.03 (dd, J=12.6, 5.4 Hz, 1H, H—C(5)), 1.51 (ddd, J=12.7,11.2, 4.2 Hz, 1H, H—C(5)), 1.08 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 146.24 (C(2)), 135.72, 135.69 (CH-arom),134.03, 133.74 (C-arom), 129.80, 129.78, 127.73 (CH-arom), 101.84(C(3)), 84.59 (C(6a)), 76.79 (C(4)), 74.10 (C(6)), 55.56 (C(3a)), 39.38(C(5)), 26.93 (CH₃)₃—C—Si), 19.08 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₂₃H₂₉O₃Si ([M+H]⁺) 381.1880, found 381.1893.

Example 9(((3aR,4R,6R,6aS)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-4-yl)oxy)(tert-butyl)diphenylsilane(9)

To a solution of glycal 8 (1.34 g, 3.52 mmol) and DMTr-Cl (1.43 g, 4.23mmol) in a mixture of dry DCM (15 mL) and dry 2,6-lutidine (15 mL) wasadded portionwise silver triflate (1.13 g, 4.40 mmol), resulting in adeep red suspension. After stirring for 2 hours at rt, an additionalportion of DMTr-Cl (239 mg, 0.705 mmol) was added. The suspension wasfurther stirred for 2 hours and then was filtered. The organic phase waswashed with satd NaHCO₃ (100 mL) and aqueous phase were extracted withDCM (3×30 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC(EtOAc/hexane 1:7, +0.5% Et₃N) to yield the protected glycal 9 (2.24,93%) as a white foam.

Data for 9: R_(f)=0.59 (EtOAc/hexane 1:2);

¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J=7.4 Hz, 2H, H-arom), 7.69-7.60 (m,J=9.3, 5.9, 4.6 Hz, 8H, H-arom), 7.56-7.39 (m, 8H, H-arom), 7.33 (t,J=7.3 Hz, 1H, H-arom), 7.00-6.93 (m, 4H, H-arom), 6.47-6.37 (m, 1H,H—C(2)), 4.67-4.58 (m, 1H, H—C(6)), 4.58-4.50 (m, 2H, H—C(3), H—C(6a)),3.86, 3.85 (2s, 6H, MeO), 3.82 (d, J=4.0 Hz, 1H, H—C(4)), 3.08 (d, J=8.1Hz, 1H, H—C(3a)), 1.67 (td, J=12.4, 4.2 Hz, 1H, H—C(5)), 1.28 (dd,J=12.7, 5.4 Hz, 1H, H—C(5)), 1.11 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 158.67 (MeO—C-arom), 147.61 (C(2)), 146.26,137.36, 137.21 (C-arom), 135.81, 135.78 (CH-arom), 134.17, 134.04(C-arom), 130.48, 129.83, 129.81, 128.37, 127.98, 127.76, 127.73,126.79, 113.32, 113.28 (CH-arom), 100.29 (C(3)), 86.96 (C(Ph)₃), 84.95(C(6a)), 76.17 (C(6)), 76.07 (C(4)), 55.26 (MeO-DMTr), 55.11 (C(3a)),37.32 (C(5)), 27.04 (CH₃)₃—C—Si), 19.21 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₄₄H₄₆O₅NaSi ([M+Na]⁺) 705.3007, found 705.3021.

Example 10(3aS,4R,6R,6aS)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-4-ol(10)

To a solution of glycal 9 (2.23 g, 3.27 mmol) in dry THF (20 mL) wasadded TBAF (1M in THF, 20 mL, 20 mmol) at rt. The solution was stirredfor 20 h and then was diluted with satd NaHCO₃ (100 mL) and extractedwith DCM (3×80 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC (0.5% MeOHin DCM, +0.5% Et₃N) to yield 10 (1.45 g, quant.) as a white foam.

Data for 10: R_(f)=0.44 (EtOAc/hexane 1:1);

¹H NMR (300 MHz, CDCl₃) δ 7.53-7.46 (m, 2H, H-arom), 7.43-7.35 (m, 4H,H-arom), 7.21 (dd, J=10.7, 5.3 Hz, 2H, H-arom), 7.16-7.08 (m, 1H,H-arom), 6.80-6.71 (m, 4H, H-arom), 6.30 (t, J=2.1 Hz, 1H, H—C(2)), 4.68(t, J=2.8 Hz, 1H, H—C(3)), 4.29-4.14 (m, 2H, H—C(6), H—C(6a)), 3.71 (s,6H, MeO), 3.65 (d, J=3.5 Hz, 1H, H—C(4)), 2.87 (d, J=7.9 Hz, 1H,H—C(3a)), 1.59 (ddd, J=13.2, 11.6, 4.3 Hz, 1H, H—C(5)), 1.05-0.95 (m,2H, H—C(5), OH).

¹³C NMR (75 MHz, CDCl₃) δ 158.54 (MeO—C-arom), 147.64 (C(2)), 145.82,137.12, 137.08 (C-arom), 130.26, 128.29, 127.81, 126.71, 113.13(CH-arom), 100.17 (C(3)), 86.75 (C(Ph)₃), 84.42 C(6a)), 75.54 (C(6)),74.59 (C(4)), 55.22 (MeO-DMTr), 54.25 (C(3a)), 37.56 (C(5)).

ESI⁺-HRMS m/z calcd for C₃₀H₂₇O₅ ([M+H]⁺) 467.1853, found 467.1844.

Example 11(3′S,5′R,7′R)-1-{2′,3′-Dideoxy-3′,5′-ethano-7′-hydroxy-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}thymine (11)

To a solution of glycal 10 (1.45 g, 3.27 mmol) in dry DCM (45 mL), at0°, was added dropwise BSA (2.0 mL, 8.18 mmol) and then the solution wasallowed to warm to rt. After stirring for 45 min, Thymine (595 mg, 4.91mmol) was added and the reaction was further stirred for 60 min at rt.The mixture was then cooled down to 0° C. and N-iodosuccinimide (875 mg,3.92 mmol) was added. After stirring for 3 h at 0° C. and for 4 h at rt,the reaction mixture was diluted with EtOAc (100 mL) and subsequentlywashed with a 10% aq solution of Na₂S₂O₃ (100 mL) and satd NaHCO₃ (100mL). Aqueous phases were combined and extracted with DCM (3×50 mL). Thecombined organic phases were dried over MgSO₄, filtered and evaporated.

The crude product was dissolved in dry toluene (45 mL) and then Bu₃SnH(1.32 mL, 4.91 mmol) and azoisobutyronitrile (AIBN, 53 mg, 0.33 mmol)were added at rt. After heating at 70° C. for 30 min, the mixture wascool down to rt and TBAF was added (1M in THF, 6.5 mL, 6.5 mmol). Thesolution was further stirred for 25 min and was diluted with satd NaHCO₃(100 mL) and extracted with DCM (4×70 mL). The combined organic phaseswere dried over MgSO₄, filtered and evaporated. The crude product waspurified by CC (3% MeOH in DCM, +0.5% Et₃N) to yield 11 (1.45 g, 73%over two steps) as a white foam.

Data for 11: R_(f)=0.29 (6% MeOH in DCM);

¹H NMR (400 MHz, CDCl₃) δ 9.37 (br, 1H, H—N(3)), 7.83 (d, J=1.1 Hz, 1H,H—C(6)), 7.58-7.52 (m, 2H, H-arom), 7.48-7.41 (m, 4H, H-arom), 7.28 (t,J=7.7 Hz, 2H, H-arom), 7.21 (t, J=7.2 Hz, 1H, H-arom), 6.84 (dd, J=8.9,1.2 Hz, 4H, H-arom), 5.91 (dd, J=8.0, 5.5 Hz, 1H, H—C(1′)), 4.25 (dt,J=10.8, 6.0 Hz, 1H, H—C(5′)), 4.13-4.08 (m, 1H, H—C(4′)), 3.86 (d, J=3.4Hz, 1H, H—C(7′), 3.79 (s, 6H, MeO), 2.70 (ddd, J=12.8, 10.2, 5.5 Hz, 1H,H—C(2′)), 2.61 (dd, J=16.9, 8.2 Hz, 1H, H—C(3′)), 1.84 (d, J=0.8 Hz, 3H,Me-C(5)), 1.80 (br, 1H, OH), 1.60 (ddd, J=14.2, 10.5, 4.2 Hz, 1H,H—C(6′)), 1.33 (dt, J=12.9, 8.0 Hz, 1H, H—C(2′)), 1.14 (dd, J=13.7, 6.1Hz, 1H, H—C(6′)).

¹³C NMR (101 MHz, CDCl₃) δ 164.17 (C(4)), 158.64 (MeO—C-arom), 150.47(C(2)), 145.65, 136.85, 136.71 (C-arom), 135.52 (C(6)), 130.20, 128.12,127.91, 126.90, 113.22, 113.21 (CH-arom), 110.69 (C(5)), 87.21 (C(Ph)₃),86.57 (C(1′)), 82.02 (C(4′)), 74.19 (C(5′)), 74.13 (C(7′)), 55.25(MeO-DMTr), 49.40 (C(3′)), 38.51 (C(6′)), 37.64 (C(2′)), 12.58(Me-C(5)).

ESI⁺-HRMS m/z calcd for C₃₃H₃₄O₇N₂Na ([M+NA]⁺) 593.2258, found 593.2250.

Example 12(3′R,5′R,7′R)-1-{7′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2′,3′-Dideoxy-3′,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}Thymine (12)

To a solution of the nucleoside 11 (232 mg, 0.406 mmol) and5-(Ethylthio)-1H-tetrazole (90 mg, 0.69 mmol) in dry DCM (10 mL) wasadded dropwise 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(0.26 mL, 0.81 mmol) at rt. After stirring for 30 min, the reactionmixture was diluted with DCM (50 mL) and washed with satd NaHCO₃ (2×30mL) and satd NaCl (30 mL). Aqueous phases were combined and extractedwith DCM (50 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC (1.8% MeOHin DCM, +0.5% Et₃N) to yield 12 (219 mg, mixture of two isomers, 70%) asa white foam.

Data for 11: R_(f)=0.68 (6% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 8.93 (br, 1H, H—N(3)), 7.85 (d, J=1.2 Hz, 1H,H—C(6)), 7.65-7.52 (m, 2H, H-arom), 7.52-7.40 (m, 4H, H-arom), 7.40-7.21(m, 3H, H-arom), 6.96-6.81 (m, 4H, H-arom), 6.00, 5.94 (2dd, J=8.3, 5.2Hz, 1H, H—C(1′)), 4.29-4.17 (m, 1H, H—C(5′)), 4.12-3.89 (m, 2H, H—C(4′),H—C(7′)), 3.85, 3.84 (2s, 6H, MeO), 3.81-3.63 (m, 2H, OCH₂CH₂CN),3.56-3.41 (m, 2H, (Me₂CH)₂N), 2.88-2.69 (m, 2H, H—C(3′), H—C(2′)), 2.61,2.56 (dt, J=12.9 6.3 Hz, 2H, OCH₂CH₂CN), 1.92, 1.82 (2d, J=0.8 Hz, 3H,Me-C(5)), 1.75-1.56 (m, 1H, H—C(6′)), 1.52-1.36 (m, 2H, H—C(6′),H—C(2′)), 1.22-1.01 (m, 12H, (Me₂CH)₂N).

¹³C NMR (101 MHz, CDCl₃) δ 163.86 (C(4)), 158.66, 158.64 (MeO—C-arom),150.29, 150.27 (C(2)), 145.58, 145.52, 136.76, 136.71, 136.69, 136.60(C-arom), 135.49, 135.35 (C(6)), 130.21, 130.16, 128.17, 128.13, 127.88,126.91, 126.89 (CH-arom), 117.49 (OCH₂CH₂CN), 113.18 (CH-arom), 110.74(C(5)), 87.27, 87.25 (C(Ph)₃), 86.58, 86.45 (C(1′)), 81.79, 81.68(C(4′)), 76.02, 75.50 (J_(C,P)=16.5, 15.7 Hz, C(7′)), 74.22 (C(5′)),58.26, 58.06, 57.87 (OCH₂CH₂CN), 55.26, 55.22 (MeO-DMTr), 48.85, 48.62(J_(C,P)=2.6, 5.0 Hz, C(3′)), 43.10, 43.04 (J_(C,P)=12.3, 12.4 Hz(Me₂CH)₂N), 37.78 (J_(C,P)=5.3 Hz C(6′)), 37.62, 37.48 (C(2′)), 37.41(J_(C,P)=3.6 Hz C(6′)), 24.57, 24.53, 24.50, 24.46, 24.44, 24.39, 24.37(Me₂CH)₂N), 20.35, 20.25 (J_(C,P)=7.1, 7.0 Hz, OCH₂CH₂CN), 12.58, 12.41(7s, Me-C(5)).

³¹P NMR (122 MHz, CDCl₃) δ 147.32, 146.98.

ESI⁺-HRMS m/z calcd for C₄₂H₅₂O₈N₄P ([M+H]⁺) 771.3517, found 771.3512.

Example 13(3'S,5′R,7′R)—N4-Benzoyl-1-{2′,3′-Dideoxy-3′,5′-ethano-7′-hydroxy-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}-5-methylcytosine(13)

To a solution of the nucleoside 11 (302 mg, 0.530 mmol) in dry MeCN (5mL) was added dropwise BSA (0.31 mL, 1.27 mmol) at 0°, and then thesolution was stirred overnight at rt. In another flask, a suspension of1,2,4-triazole (1.28 g, 18.55 mmol) in dry MeCN (50 mL) was cool down to0° C. and POCl₃ (0.40 mL, 4.2 mmol) and Et₃N (2.96 mL, 21.2 mmol) wereadded. The suspension was stirred for 30 min at 0° C., and then theprevious prepared solution of the silylated compound 11 was added to thesuspension and the mixture was further stirred for 5 h at rt. Reactionwas quenched with addition satd NaHCO₃ (10 mL), MeCN removed underreduced pressure and the resulting mixture diluted with satd NaHCO₃ (35mL) and extracted with DCM (3×40 mL). The combined organic phases weredried over MgSO₄, filtered and evaporated.

The crude product was then dissolved in a mixture of 1,4-dioxane (10 mL)and concd NH₄OH (10 mL). After stirring for 2 h at rt, the mixture wasreduced to half of the volume in vacuo, diluted with satd NaHCO₃ (30 mL)and extracted with DCM (4×30 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated.

The crude product was then dissolved in dry DMF (13 mL), Et₃N (90 μL,0.64 mmol) followed by Bz₂O (300 mg, 1.33 mmol) were added at rt and thesolution was stirred overnight. The resulting brown solution wasquenched by careful addition of satd NaHCO₃ (50 mL) and extracted withDCM (4×50 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC(hexane/EtOAc 1:2, +0.5% Et₃N) to yield 13 (315 mg, 88%) as a whitefoam.

Data for 13: R_(f)=0.57 (4% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 13.39 (br, 1H, NH), 8.46-8.26 (m, 2H, H-arom),8.13 (d, J=0.5 Hz, 1H, C(6)), 7.61 (d, J=7.3 Hz, 2H, H-arom), 7.58-7.43(m, 7H, H-arom), 7.34 (t, J=7.4 Hz, 2H, H-arom), 7.30-7.23 (m, 1H,H-arom), 6.89 (d, J=8.8 Hz, 4H, H-arom), 5.96 (dd, J=7.5, 5.8 Hz, 1H,H—C(1′)), 4.38-4.25 (m, 1H, H—C(5′)), 4.22-4.12 (m, 1H, H—C(4′)), 3.90(d, J=3.6 Hz, 1H, H—C(7′)), 3.83 (s, 6H, MeO), 2.82 (ddd, J=13.3, 10.2,5.7 Hz, 1H, H—C(2′)), 2.66 (dd, J=17.0, 8.1 Hz, 1H, H—C(3′)), 2.08 (s,3H, Me-C(5)), 1.77 (br, 1H, OH), 1.71-1.57 (m, 1H, H—C(6′)), 1.49-1.36(m, 1H, H—C(2′)), 1.21 (dd, J=13.7, 6.2 Hz, 1H, H—C(6′)).

¹³C NMR (75 MHz, CDCl₃) δ 179.56 (CONH), 160.01 (C(4)), 158.70(MeO—C-arom), 147.96 (C(2)), 145.65 (C-arom), 137.26 (C(6)), 136.99,136.83, 136.71 (C-arom), 132.41, 130.22, 129.89, 128.16, 128.14, 127.95,126.94, 113.25 (CH-arom), 111.57 (C(5)), 87.34 (C(Ph)₃), 87.32 (C(1′)),82.57 (C(4′)), 74.30 (C(5′)), 74.16 (C(7′)), 55.27 (MeO-DMTr), 49.56(C(3′)), 38.52 (C(6′)), 38.00 (C(2′)), 13.63 (Me-C(5)).

ESI⁺-HRMS m/z calcd for C₄₀H₄₀O₇N₃ ([M+H]⁺) 674.2861, found 674.2862.

Example 14(3′R,5′R,7′R)—N4-Benzoyl-1-{7′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2′,3′-Dideoxy-3′,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}-5-methylcytosine(14)

To a solution of the nucleoside 13 (276 mg, 0.409 mmol) and5-(Ethylthio)-1H-tetrazole (69 mg, 0.53 mmol) in dry DCM (10 mL) wasadded dropwise 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(0.20 mL, 0.61 mmol) at rt. After stirring for 60 min, the reactionmixture was diluted with DCM (50 mL) and washed with satd NaHCO₃ (2×30mL) and satd NaCl (30 mL). Aqueous phases were combined and extractedwith DCM (50 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC(EtOAc/Hexanne 2:3, +0.5% Et₃N) to yield 14 (268 mg, mixture of twoisomers, 75%) as a white foam.

Data for 14: R_(f)=0.77 (5% MeOH in DCM);

¹H NMR (400 MHz, CDCl₃) δ 13.32 (s, 1H, NH), 8.41-8.28 (m, 2H, H-arom),8.13-8.04 (m, 1H, C(6)), 7.61-7.51 (m, 3H, H-arom), 7.51-7.40 (m, 6H,H-arom), 7.37-7.29 (m, 2H, H-arom), 7.29-7.20 (m, 1H, H-arom), 6.92-6.82(m, 4H, H-arom), 6.07-5.87 (m, 1H, H—C(1′)), 4.24 (dq, J=11.7, 5.8 Hz,1H, H—C(5′)), 4.13-4.00 (m, 1H, H—C(4′)), 3.94 (ddd, J=14.5, 10.5, 2.8Hz, 1H, H—C(7′)), 3.83, 3.82 (2s, 6H, MeO), 3.69 (m, 2H, OCH₂CH₂CN),3.53-3.40 (m, 2H, (Me₂CH)₂N), 2.91-2.70 (m, 2H, H—C(2′), H—C(3′)), 2.57,2.53 (2t, J=6.3 Hz, 2H, OCH₂CH₂CN), 2.08, 1.99 (2d, J=0.6 Hz, 3H,Me-C(5)), 1.72 1.56 (m, 1H, H—C(6′)), 1.54-1.36 (m, 2H, H—C(2′),H—C(6′)), 1.10 (m, 12H, (Me₂CH)₂N).

¹³C NMR (101 MHz, CDCl₃) δ 179.54 (CONH), 159.98 (C(4)), 158.69(MeO—C-arom), 147.90 (C(2)), 145.58, 145.54 (C-arom), 137.30, 136.93(C(6)), 136.81, 136.80, 136.73, 136.70, 136.67, 136.60 (C-arom), 132.37,132.35, 130.22, 130.17, 129.89, 128.17, 128.15, 128.11, 127.93, 126.94(CH-arom), 117.49 (OCH₂CH₂CN), 113.23 (CH-arom), 111.60 (C(5)), 87.36,87.35 (C(Ph)₃), 87.33, 87.25 (C(1′)), 82.33, 82.25 (C(4′)), 76.05, 75.52(J_(C,P)=16.4, 15.6 Hz, C(7′)), 74.32 (C(5′)), 58.18, 57.98(J_(C,P)=19.5 Hz OCH₂CH₂CN), 55.28, 55.24 (MeO-DMTr), 48.93, 48.72(J_(C,P)=2.7, 4.9 Hz, C(3′)), 43.11, 43.05 (J_(C,P)=12.4 Hz (Me₂CH)₂N),38.02, 37.88 (C(2′)), 37.74, 37.40 (J_(C,P)=5.3, 3.4 Hz, C(6′)), 24.58,24.54, 24.50, 24.47, 24.40, 24.38 (6s, Me₂CH)₂N), 20.36, 20.26(J_(C,P)=7.1 Hz, OCH₂CH₂CN),), 13.66, 13.49 (Me-C(5)).

³¹P NMR (122 MHz, CDCl₃) δ 147.37, 147.07.

ESI⁺-HRMS m/z calcd for C₄₉H₅₇O₈N₅P ([M+H]⁺) 874.3939, found 874.3937.

Example 15(3′R,5′R,7′R)—N6-Benzoyl-9-{5′-O-acetyl-7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-α,β-D-ribofuranosyl}adenine (15)

To a suspension of sugar 7 (1.86 g, 4.10 mmol) and N⁶-Benzoyladenine(1.96 g, 8.20 mmol) in dry MeCN (40 mL) was added BSA (4.00 mL, 16.4mmol) at rt. After stirring for 25 min, the suspension became a clearsolution and then was heated to 70° C. TMSOTf (1.48 mL, 8.20 mmol) wasadded dropwise and the solution was further stirred for 20 min at 70° C.The solution was then cool down to rt, quenched with addition of satdNaHCO₃ (100 mL) and extracted with EtOAc (4×50 mL). The combined organicphases were dried over MgSO₄, filtered and evaporated. The crude productwas purified by CC (2% MeOH in DCM) to yield a mixture of 15 (1.74 g,64%) in an anomeric ratio α/β≈4:1 as a white foam.

Data for 15: R_(f)=0.33 (EtOAc/hexane 4:1);

¹H NMR (400 MHz, CDCl₃) δ 9.33 (br, 1H, NH), 8.68 (d, J=5.4 Hz, 0.8H,H—C(2)), 8.64 (d, J=5.6 Hz, 0.2H, H—C(2)), 8.10 (d, J=1.5 Hz, 0.2H.H—C(8)), 7.99 (d, J=7.3 Hz, 2H, H-arom), 7.95 (s, 0.8H, H—C(8)), 7.63(t, J=8.7 Hz, 4H, H-arom), 7.55 (dd, J=13.0, 6.4 Hz, 1H, H-arom),7.50-7.34 (m, 8H, H-arom), 6.20 (dd, J=6.3, 2.5 Hz, 0.8H, H—C(1′)), 6.05(t, J=6.5 Hz, 0.2H, H—C(1′)), 5.43-5.32 (m, 1H, H—C(5′)), 5.03-4.97 (m,0.8H, H—C(4′)), 4.83 (t, J=6.0 Hz, 0.2H, H—C(4′)), 4.14 (br, 0.2H,H—C(7′)), 4.08 (d, J=3.7 Hz, 0.8H, H—C(7′)), 3.02 (dd, J=16.1, 6.6 Hz,0.8H, H—C(3′)), 2.83 (dd, J=16.9, 7.7 Hz, 0.2H, H—C(3′)), 2.59-2.39 (m,1H, H—C(2′)), 2.18-2.11 (m, 1H, H—C(6′)), 2.07 (d, J=1.6 Hz, 2.4H,MeCO₂), 2.02 (d, J=1.9 Hz, 0.6H, MeCO₂), 2.01-1.92 (m, 1H, H—C(6′)),1.91-1.80 (m, 1H, H—C(3′)), 1.07 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (101 MHz, CDCl₃) δ 170.57, 170.49 (MeCO₂), 164.82 (CONH), 152.50(C(2)), 151.27 (C(4)), 149.56 (C(6)), 141.37, 141.06 (C(8)), 135.72,135.68, 135.66 (CH-arom), 133.67, 133.57, 133.24, 133.22 (C-arom),132.73, 130.03, 129.98, 128.80, 128.78, 127.92, 127.86, 127.85(CH-arom), 123.61 (C(5)), 87.19, 86.17 (C(1′)), 83.22, 80.96 (C(4′),76.50, 76.04 (C(7′)), 74.38 (C(5′)), 51.07 (C(3′)), 37.29, 37.15, 36.80,36.60 (C(2′), C(6′)), 26.89 (CH₃)₃—C—Si), 20.97, 20.90 (MeCO₂), 19.01(CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₃₇H₄₀O₅N₅Si ([M+H]⁺) 662.2793, found 662.2787.

Example 16(3′R,5′R,7′R)—N6-Benzoyl-9-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-β-D-ribofuranosyl}adenine (16)

The nucleoside 15 (1.74 g, 2.64 mmol) was dissolved in 0.15 M NaOH inTHF/methanol/H₂O (5:4:1, 80 mL) at 0° C. The reaction was stirred for 20min and quenched by addition of NH₄Cl (1.06 g). Solvents were thenremoved under reduced pressure and the product purified by CC (5%isopropanol in DCM) to yield 16 (287 mg, 18%) and its corresponding αanomer (836 mg, 51%) white foams.

Data for 16: R_(f)=0.44 (6% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 8.70 (s, 1H, H—C(2)), 8.09-7.98 (m, 2H,H-arom), 7.97 (s, 1H, H—C(8)), 7.63 (ddd, J=7.4, 5.7, 1.5 Hz, 4H,H-arom), 7.59-7.55 (m, 1H, H-arom), 7.51 (m, 2H, H-arom), 7.44-7.33 (m,6H, H-arom), 6.02 (dd, J=9.4, 5.5 Hz, 1H, H—C(1′)), 4.57 (dd, J=8.1, 5.0Hz, 1H, H—C(4′)), 4.43 (dd, J=11.8, 5.3 Hz, 1H, H—C(5′)), 4.26 (br, 1H,H—C(7′)), 2.78 (q, J=8.9 Hz, 1H, H—C(3′)), 2.32-1.80 (m, 5H, H—C(2′),H—C(6′), OH), 1.06 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (101 MHz, CDCl₃) δ 164.85 (CONH), 152.56 (C(2)), 151.17 (C(4)),149.86 (C(6)), 141.25 (C(8)), 135.68 (CH-arom), 133.87, 133.39 (C-arom),132.78, 129.92, 128.78, 128.01, 127.78 (CH-arom), 123.51 (C(5)), 87.65(C(1′)), 82.91 (C(4′)), 76.66 (C(7′)), 72.54 (C(5′)), 50.44 (C(3′)),41.42 (C(6′)), 36.17 (C(2′)), 26.89 (CH₃)₃—C—Si), 19.03 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₃₅H₃₈O₄N₅Si ([M+H]⁺) 620.2688, found 620.2671.

Example 17(3′R,5′R,7′R)—N6-Benzoyl-9-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}adenine (17)

To a solution of nucleoside 16 (307 mg, 0.495 mmol) in dry pyridine (6mL) was added DMTr-Cl (503 mg, 1.49 mmol) at rt. The solution wasstirred for 1 day and then diluted with satd NaHCO₃ (50 mL) andextracted with DCM (3×70 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (1.5% MeOH in DCM, +0.5% Et₃N) to yield 17 (395 mg, 87%) as a yellowfoam.

Data for 17: R_(f)=0.65 (5% MeOH in DCM);

¹H NMR (300 MHz, MeOD) δ 8.64 (s, 1H, H—C(2)), 8.61 (s, 1H, H—C(8)),8.08 (d, J=7.2 Hz, 2H, H-arom), 7.68-7.17 (m, 22H, H-arom), 6.86-6.75(m, 4H, H-arom), 6.14 (dd, J=7.4, 6.3 Hz, 1H, H—C(1′)), 4.48-4.31 (m,1H, H—C(5′)), 4.28-4.15 (m, 1H, H—C(4′)), 3.88 (d, J=3.8 Hz, 1H,H—C(7′)), 3.75, 3.74 (2s, 6H, MeO), 2.67 (dd, J=16.6, 6.7 Hz, 1H,H—C(3′)), 2.47 (ddd, J=13.3, 10.2, 6.1 Hz, 1H, H—C(2′)), 2.15-1.94 (m,1H, H—C(6′)), 1.71 (ddd, J=13.0, 11.3, 4.4 Hz, 1H, H—C(2′)), 1.11 (dd,J=12.2, 4.9 Hz, 1H, H—C(6′)), 0.95 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 164.69 (CONH), 158.61, 158.60 (MeO—C-arom),152.42 (C(2)), 151.27 (C(4)), 149.41 (C(6)), 145.81 (C-arom), 141.25(C(8)), 137.00, 136.85 (C-arom), 135.60, 135.57 (CH-arom), 133.80,133.69, 133.43 (C-arom), 132.70, 130.28, 130.25, 129.85, 129.81, 128.84,128.18, 127.89, 127.71, 127.65, 126.78 (CH-arom), 123.52 (C(5)), 113.22,113.19 (CH-arom), 87.09 (C(Ph)₃), 86.41 (C(1′)), 83.52 (C(4′)), 76.05(C(7′)), 74.78 (C(5′)), 55.20 (MeO-DMTr), 50.43 (C(3′)), 38.10 (C(2′),C(6′)), 26.84 (CH₃)₃—C—Si), 19.00 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₅₆H₅₆O₆N₅Si ([M+H]⁺) 922.3994, found 922.3953.

Example 18(3′S,5′R,7′R)—N6-Benzoyl-9-{2′,3′-Dideoxy-3′,5′-ethano-7′-hydroxy-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}adenine (18)

To a solution of nucleoside 17 (376 mg, 0.408 mmol) in dry THF (9 mL)was added TBAF (1M in THF, 1.22 mL, 1.22 mmol) at rt. The solution wasstirred for 2 days and was then diluted with satd NaHCO₃ (25 mL) andextracted with DCM (4×25 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (4% MeOH in DCM, +0.5% Et₃N) to yield 18 (242 mg, 87%) as a whitefoam.

Data for 18: R_(f)=0.33 (5% MeOH in DCM);

¹H NMR (300 MHz, CD₃CN) δ 9.35 (br, 1H, NH), 8.67 (s, 1H, C(2)), 8.46(s, 1H, C(8)), 8.01 (d, J=7.4 Hz, 2H, H-arom), 7.54 (m, 5H, H-arom),7.35 (m, 4H, H-arom), 7.30-7.17 (m, 3H, H-arom), 6.84 (d, J=8.9 Hz, 4H,H-arom), 6.09 (dd, J=7.8, 6.2 Hz, 1H, H—C(1′)), 4.12 (dt, J=11.2, 5.8Hz, 1H, C(5′)), 3.87-3.79 (m, 2H, C(4′), C(7′)), 3.75 (s, 6H, MeO),2.83-2.64 (m, 2H, C(2′), OH), 2.58-2.46 (m, 1H, C(3′)), 2.21 (dd,J=13.9, 7.1 Hz, 1H, C(2′)), 1.92-1.82 (m, 1H, C(6′)), 1.29-1.17 (m, 1H,C(6′)).

¹³C NMR (75 MHz, CDCl₃) δ 165.03 (CONH), 158.57 (MeO—C-arom), 152.40(C(2)), 151.23 (C(4)), 149.52 (C(6)), 145.68 (C-arom), 141.49 (C(8)),136.86, 136.84, 133.77 (C-arom), 132.77, 130.22, 128.81, 128.16, 128.02,127.89, 126.84 (CH-arom), 123.40 (C(5)), 113.19 (CH-arom), 87.06(C(Ph)₃), 86.74 (C(1′)), 83.58 (C(4′)), 74.62 (C(5′)), 74.38 (C(8′)),55.25 (MeO-DMTr), 49.77 (C(3′)), 38.55, 38.32 (C(6′), C(2′)).

ESI⁺-HRMS m/z calcd for C₄₀H₃₈O₆N₅ ([M+H]⁺) 684.2817, found 684.2830.

Example 19(3′R,5′R,7′R)—N6-Benzoyl-9-{7′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2′,3′-Dideoxy-3′,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}Adenine (19)

To a solution of the nucleoside 18 (173 mg, 0.253 mmol) andN,N-Diisopropylethylamine (0.18 mL, 1.0 mmol) in dry THF (8 mL) wasadded N,N-diisopropylchlorophosphoramidite (0.11 mL, 0.50 mmol) at rt.The solution was stirred for 2 hours and then was diluted with satdNaHCO₃ (40 mL) and extracted with DCM (4×40 mL). The combined organicphases were dried over MgSO₄, filtered and evaporated. The crude productwas purified by CC (EtOAc, +0.5% Et₃N) to yield 19 (177 mg, mixture oftwo isomers, 71%) as a white foam.

Data for 19: R_(f)=0.38, 0.44 (EtOAc);

¹H NMR (400 MHz, CDCl₃) δ 9.05 (br, 1H, NH), 8.70, 8.70 (2s, 1H,H—C(2)), 8.47, 8.46 (2s, 1H, H—C(8)), 7.97 (d, J=7.5 Hz, 2H, H-arom),7.57-7.50 (m, 1H, H-arom), 7.49-7.41 (m, 4H, H-arom), 7.39-7.31 (m, 4H,H-arom), 7.24-7.17 (m, 5.4 Hz, 2H, H-arom), 7.13 (dt, J=12.5, 6.2 Hz,1H, H-arom), 6.83-6.70 (m, 4H, H-arom), 6.14-5.97 (m, 1H, H—C(1′)), 4.14(ddd, J=11.1, 7.8, 3.4 Hz, 1H, H—C(5′)), 3.91-3.74 (m, 2H, H-(4′),H—C(7′)), 3.71, 3.70 (2s, 6H, MeO), 3.65-3.50 (m, 2H, OCH₂CH₂CN), 3.37(ddq, J=13.9, 10.2, 6.8 Hz, 2H, (Me₂CH)₂N), 2.90-2.76 (m, 1H, H—C(2′)),2.75-2.60 (m, 1H, H—C(3′)), 2.47, 2.42 (2t, J=6.3 Hz, 2H, OCH₂CH₂CN),2.11 (dt, J=12.7, 6.1 Hz, 1H, H—C(2′)), 1.73 (ddt, J=13.6, 10.4, 5.1 Hz,1H, H—C(6′)), 1.39 (ddd, J=50.2, 13.4, 6.2 Hz, 1H, H—C(6′)), 1.10-0.89(m, 12H, (Me₂CH)₂N).

¹³C NMR (101 MHz, CDCl₃) δ 164.66 (CONH), 158.57 (MeO—C-arom), 152.46(C(2)), 151.32, 151.26 (C(4)), 149.45, 149.43 (C(6)), 145.60, 145.59(C-arom), 141.52, 141.47 (C(8)), 136.88, 136.83, 136.81, 133.78(C-arom), 132.75, 132.73, 130.22, 130.21, 130.19, 130.17, 128.87,128.17, 127.87, 126.82, 126.80 (CH-arom), 123.59 (C(5)), 117.53, 117.50(OCH₂CH₂CN), 113.17 (CH-arom), 87.10, 87.07 (C(Ph)₃), 86.72, 86.68(C(1′)), 83.36, 83.25 (C(4′)), 76.55, 75.81 (J_(C,P)=16.9, 15.7 Hz,C(7′)), 74.63, 74.60 (C(5′)), 58.24, 57.86 (J_(C,P)=19.1, 19.2 HzOCH₂CH₂CN), 55.25, 55.21 (MeO-DMTr), 49.29, 49.08 (J_(C,P)=2.6, 4.7 Hz,C(3′)), 43.12, 43.00 (J_(C,P)=2.4, 2.3 Hz (Me₂CH)₂N), 38.27, 38.23(C(2′)), 37.41, 37.22 (J_(C,P)=5.3, 3.5 Hz, C(6′)) 24.56, 24.53, 24.49,24.47, 24.43, 24.41, 24.36, 24.33 (8s, Me₂CH)₂N), 20.36, 20.25(J_(C,P)=7.2, 7.0 Hz, OCH₂CH₂CN).

³¹P NMR (122 MHz, CDCl₃) δ 147.64, 146.87.

ESI⁺-HRMS m/z calcd for C₄₉H₅₅O₇N₇ ([M+H]⁺) 884.3895, found 884.3898.

Example 20(3′R,5′R,7′R)-2-Amino-6-chloro-9-{5′-O-acetyl-7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-α,β-D-ribofuranosyl}purine (20)

To a suspension of sugar 7 (1.75 g, 3.85 mmol) and2-amino-6-chloropurine (1.05 g, 6.17 mmol) in dry MeCN (20 mL) was addedBSA (3.80 mL, 15.4 mmol) at rt. The suspension was heated to 55° C. andstirred for 30 min. Then TMSOTf (1.05 mL, 5.78 mmol) was added dropwiseand the solution was further stirred for 50 min at 55° C. The solutionwas cool down to rt, quenched with addition of satd NaHCO₃ (10 mL),diluted EtOAc (50 mL) and filtered through a short pad of SiO₂. The SiO₂was washed with additional EtOAc. The mixture was then washed with satdNaHCO₃ (2×80 mL), aqueous phases were combined and extracted with EtOAc(3×50 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (2.5% MeOH in DCM)to yield a mixture of 20 (1.77 g, 77%) in an anomeric ratio α/β≈7:3 as awhite foam.

Data for 20: R_(f)=0.54 (EtOAc/hexane 5:1);

¹H NMR (300 MHz, CDCl₃) δ 7.86 (s, 0.3H, H—C(8)), 7.69 (s, 0.7H,H—C(8)), 7.68-7.60 (m, 4H, H-arom), 7.47-7.34 (m, 6H, H-arom), 6.04 (dd,J=6.9, 3.0 Hz, 0.7H, H—C(1′)), 5.87 (dd, J=8.0, 6.2 Hz, 0.3H, H—C(1′)),5.37 (dt, J=14.2, 4.6 Hz, 1H, H—C(5′)), 5.16 (br, 2H, NH₂), 4.91 (dd,J=6.5, 5.1 Hz, 0.7H, H—C(4′)), 4.79 (dd, J=6.9, 5.2 Hz, 0.3H, H—C(4′)),4.13 (br, 0.3H, H—C(7′)), 4.06 (d, J=4.0 Hz, 0.7H, H—C(7′)), 2.95 (dd,J=16.3, 6.6 Hz, 0.7H, H—C(3′)), 2.81 (dd, J=17.0, 7.4 Hz, 0.3H,H—C(3′)), 2.49-2.30 (m, 1H, H—C(2′)), 2.14 (dd, J=13.1, 6.7 Hz, 1H,H—C(6′)), 2.08 (s, 2.1H, MeCO₂), 2.02 (s, 0.9H, MeCO₂), 2.02-1.91 (m,1H, H—C(6′)), 1.80 (td, J=13.4, 6.8 Hz, 1H, H—C(2′)), 1.07, 1.06 (2s,9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 170.55, 170.44 (MeCO₂), 158.98, 158.91 (C(2)),153.18, 152.95 (C(4)), 151.40, 151.34 (C(6)), 140.38, 140.14 (C(8)),135.73, 135.70 (CH-arom), 133.78, 133.62, 133.24, 133.17 (C-arom),130.03, 130.00, 127.88, 127.86 (CH-arom), 125.65, 125.57 (C(5)), 86.59,85.74 (C(1′)), 82.93, 80.99 (C(4′)), 76.57, 76.14 (C(7′)), 74.34, 74.32(C(5′)), 51.15, 51.10 (C(3′)), 37.19, 36.99 (C(6′)), 36.70, 36.25(C(2′)), 26.87 (CH₃)₃—C—Si), 20.95, 20.86 (MeCO₂), 19.00 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₃₀H₃₅O₄N₅ClSi ([M+H]⁺) 592.2141, found592.2158.

Example 21(3′R,5′R,7′R)-2-Amino-6-chloro-9-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3,5′-ethano-β-D-ribofuranosyl}purine (22b)

The nucleoside 20 (1.78 g, 3.01 mmol) was dissolved in 0.5 M NaOH inTHF/methanol/H₂O (5:4:1, 15 mL) at 0° C. The reaction was stirred for 20min at 0° C. and quenched by addition of NH₄Cl (484 mg). The suspensionwas then diluted with satd NaHCO₃ (100 mL) and extracted with DCM (4×75mL). The combined organic phases were dried over MgSO₄, filtered andevaporated. The crude product was purified by CC (3% MeOH in DCM) toyield 21 (428 mg, 25%) and its corresponding α anomer (992 mg, 60%) aswhite foams.

Data for 21: R_(f)=0.43 (5% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 7.71 (s, 1H, H—C(8)), 7.68-7.60 (m, 4H,H-arom), 7.44-7.33 (m, 6H, H-arom), 5.85 (dd, J=9.3, 5.8 Hz, 1H,H—C(1′)), 5.33 (br, 2H, NH₂), 4.62 (dd, J=8.4, 4.9 Hz, 1H, H—C(4′)),4.44 (dd, J=10.7, 5.3 Hz, 1H, H—C(5′)), 4.40-4.15 (m, 2H, H—C(7′), OH),2.79 (q, J=8.7 Hz, 1H, H—C(3′)), 2.22 (dd, J=15.2, 9.3 Hz, 1H, H—C(6′)),2.11-2.02 (m, 1H, H—C(6′)), 2.02-1.85 (m, 2H, H—C(2′)), 1.06 (s, 9H,(CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 158.73 (C(2)), 152.78 (C(4)), 151.94 (C(6)),140.70 (C(8)), 135.70 (CH-arom), 133.91, 133.48 (C-arom), 129.90, 127.78(CH-arom), 125.97 (C(5)), 87.96 (C(1′)), 82.88 (C(5′)), 76.85 (C(7′)),72.36 (C(5′)), 50.41 (C(3′)), 41.96 (C(6′)), 35.73 (C(2′)), 26.90(CH₃)₃—C—Si), 19.02 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₂₈H₃₃O₃N₅ClSi ([M+H]⁺) 550.2036, found550.2015.

Example 22(3′R,5′R,7′R)—N2-(N,N-Dimethylformamidino)-9-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-β-D-ribofuranosyl}guanine (22)

To a solution of 21 (380 mg, 0.645 mmol) and 3-hydroxypropionitrile(0.22 mL, 3.23 mmol) in dry DCM (15 mL) was added1,5,7-Triazabicyclo[4.4.0]dec-5-ene (400 mg, 2.87 mmol) at 0° C. Thesolution was stirred for 3 hours at 0° C. and then for 2 days at rt.Reaction was stopped by addition of silica. After evaporation ofsolvent, the SiO₂ powder was filtered, washed with MeOH and solventevaporated to yield a brown foam.

The crude product was dissolved in dry DMF (5 mL) andN,N-Dimethylformamide dimethyl acetal (0.43 mL, 3.2 mmol) was added. Thesolution was stirred for 2 hours at 55° C. and then the solvents wereremoved under reduced pressure. The crude product was purified by CC (6%MeOH in DCM) to yield 23 (274 mg, 73%) as yellowish foams.

Data for 22: R_(f)=0.45 (12% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.52 (s, 1H, NH), 8.46 (s, 1H, NCHN(CH₃)₂),7.63 (dd, J=7.7, 1.5 Hz, 4H, H-arom), 7.50 (s, 1H, H—C(8)), 7.44-7.30(m, 6H, H-arom), 5.83 (dd, J=9.3, 6.0 Hz, 1H, H—C(1′)), 4.61 (dd, J=8.7,5.0 Hz, 1H, H—C(4′)), 4.43-4.32 (m, 1H, H—C(5′)), 4.29 (dd, J=7.0, 4.8Hz, 1H, H—C(7′)), 3.95 (d, J=5.1 Hz, 1H, OH), 2.98 (s, 6H, NCHN(CH₃)₂),2.79 (dd, J=18.0, 7.0 Hz, 1H, H—C(3′)), 2.20 (dt, J=12.8, 5.4 Hz, 1H,H—C(6′)), 2.09-1.88 (m, 3H, H—C(6′), H—C(2′)), 1.05 (s, 9H,(CH₃)₃—C—Si)).

¹³C NMR (75 MHz, CDCl₃) δ 158.73 (C(2)), 157.79 (C(6)), 156.91(NCHN(CH₃)₂), 149.84 (C(4)), 137.00 (C(8)), 135.70, 135.67 (CH-arom),133.78, 133.60 (C-arom), 129.93, 129.86, 127.78, 127.72 (CH-arom),121.61 (C(5)), 88.04 (C(1′)), 82.21 (C(4′)), 77.49 (C(7′)), 71.94(C(5′)), 50.13 (C(3′)), 42.23 (C(6′)), 41.20 (NCHN(CH₃)₂), 35.50(C(2′)), 34.97 (NCHN(CH₃)₂), 26.87 (CH₃)₃—C—Si), 19.02 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₃₁H₃₈O₄N₆Si ([M+H]⁺) 586.2718, found 586.2703.

Example 23(3′R,5′R,7′R)—N2-(N,N-Dimethylformamidino)-9-{7′-[(tert-butyldiphenylsilyl)oxy]-2,3′-Dideoxy-3,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (23)

To a solution of 22 (139 mg, 0.237 mmol) in dry pyridine (2 mL) wasadded DMTr-Cl (240 mg, 0.708 mmol) in six portions over 3 hours at rt.After stirring overnight, the orange solution was diluted with satdNaHCO₃ (20 mL) and extracted with DCM (3×20 mL). The combined organicphases were dried over MgSO₄, filtered and evaporated. The crude productwas purified by CC (4% MeOH in DCM, +0.5% Et₃N) to yield 23 (148 mg,70%) as yellowish foams.

Data for 23: R_(f)=0.52 (10% MeOH in DCM);

¹H NMR (400 MHz, CDCl₃) δ 9.49 (s, 1H, NH), 8.38 (s, 1H, NCHN(CH₃)₂),7.80 (s, 1H, C(8)), 7.50-7.43 (m, 2H, H-arom), 7.42-7.27 (m, 10H,H-arom), 7.26-7.15 (m, 6H, H-arom), 7.14-7.08 (m, 1H, H-arom), 6.77-6.68(m, 4H, H-arom), 5.78 (dd, J=8.2, 5.9 Hz, 1H, H—C(1′)), 4.25 (dt,J=11.0, 5.6 Hz, 1H, H—C(5′)), 4.14-4.03 (m, 1H, H—C(4′)), 3.70-3.64 (m,7H, MeO, H—C(7′)), 3.00 (s, 3H, NCHN(CH₃)₂), 2.97 (s, 3H, NCHN(CH₃)₂),2.43 (dd, J=16.7, 7.5 Hz, 1H, H—C(3′)), 2.24 (ddd, J=13.3, 10.1, 5.8 Hz,1H, H—C(2′)), 1.62 (td, J=13.1, 4.3 Hz, 1H, H—C(6′)), 1.43 (dt, J=13.5,8.0 Hz, 1H, H—C(2′)), 0.99 (dd, J=13.3, 6.2 Hz, 1H), 0.86 (s, 9H,(CH₃)₃—C—Si)).

¹³C NMR (101 MHz, CDCl₃) δ 158.51, 158.49 (MeO—C-arom), 158.04 (C(2)),157.91 (C(6)), 156.60 (NCHN(CH₃)₂), 149.76 (C(4)), 145.83, 137.12,136.94 (C-arom), 136.01 (C(8)), 135.60, 135.59 (CH-arom), 133.81, 133.47(C-arom), 130.32, 130.26, 129.77, 128.24, 127.82, 127.65, 127.62, 126.67(CH-arom), 120.65 (C(5)), 113.13, 113.09 (CH-arom), 86.82 (C(Ph)₃),85.01 (C(1′)), 82.26 (C(4′)), 76.14 (C(7′)), 74.61 (C(5′)), 55.19(MeO-DMTr), 50.18 (C(3′)), 41.29 (NCHN(CH₃)₂), 38.01 (C(6′)), 37.76(C(2′)), 35.14 (NCHN(CH₃)₂) 26.81 87 (CH₃)₃—C—Si), 19.01 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₅₂H₅₇O₆N₆Si ([M+H]⁺) 889.4103, found 889.4128.

Example 24(3'S,5′R,7′R)—N2-(N,N-Dimethylformamidino)-9-{2′,3′-Dideoxy-3′,5′-ethano-7′-hydroxy-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (24)

To a solution of 23 (243 mg, 0.273 mmol) in dry THF (2 mL) was addedTBAF (1M in THF, 1.65 mL, 1.63 mmol) at rt. The solution was stirred for7 hours and then was diluted with satd NaHCO₃ (30 mL) and extracted withDCM (4×30 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC (7% MeOHin DCM, +0.5% Et₃N) to yield 24 (155 mg, 87%) as a white foam stillcontaining traces of TBAF.

Data for 24: R_(f)=0.44 (10% MeOH in DCM);

¹H NMR (400 MHz, CDCl₃) δ 9.55 (s, 1H, NH), 8.45 (s, 1H, NCHN(CH₃)₂),8.00 (s, 1H, H—C(8)), 7.60-7.50 (m, 2H, H-arom), 7.49-7.39 (m, 4H,H-arom), 7.31-7.23 (m, 2H, H-arom), 7.21-7.12 (m, 1H, H-arom), 6.81 (d,J=8.5 Hz, 4H, H-arom), 5.93 (dd, J=7.5, 6.1 Hz, 1H, H—C(1′)), 4.26 (dt,J=11.1, 5.8 Hz, 1H, H—C(5′)), 4.07-3.98 (m, 1H, H—C(4′)), 3.91 (d, J=4.3Hz, 1H, H—C(7′)), 3.77 (s, 6H, MeO), 3.14 (s, 3H, NCHN(CH₃)₂), 3.04 (s,3H, NCHN(CH₃)₂), 2.73 (ddd, J=13.3, 10.1, 6.0 Hz, 1H, H—C(2′)),2.63-2.48 (m, 1H, H—C(3′)), 2.12 (br, 1H, OH), 1.95-1.82 (m, 2H,H—C(6′), H—C(2′)), 1.14 (dd, J=13.4, 6.1 Hz, 1H, H—C(6′)).

¹³C NMR (101 MHz, CDCl₃) δ 158.52 (MeO—C-arom), 158.12 (C(2)), 157.88(C(6)), 156.65 (NCHN(CH₃)₂), 149.78 (C(4)), 145.69, 137.02, 136.99(C-arom), 136.07 (C(8)), 130.26, 128.26, 127.82, 126.74 (CH-arom),120.53 (C(5)), 113.12 (CH-arom), 86.81 (C(Ph)₃), 85.35 (C(1′)), 82.64(C(4′)), 74.61 (C(7′)), 74.48 (C(5′)), 55.23 (MeO-DMTr), 49.63 (C(3′)),41.37 (NCHN(CH₃)₂), 38.55 (C(6′)), 38.23 (C(2′)), 35.14 (NCHN(CH₃)₂).

ESI⁺-HRMS m/z calcd for C₃₆H₃₉O₆N₆Si ([M+H]⁺) 651.2926, found 651.2912.

Example 25(3′R,5′R,7′R)—N2-(N,N-Dimethylformamidino)-9-{7′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2′,3′-Dideoxy-3,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (25)

To a solution of the nucleoside 24 (143 mg, 0.220 mmol) and5-(Ethylthio)-1H-tetrazole (43 mg, 0.33 mmol) in dry DCM (10 mL) wasadded dropwise 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(0.12 mL, 0.38 mmol) at rt. After stirring for 50 min, the reactionmixture was diluted with satd NaHCO₃ (20 mL) and extracted with DCM(3×20 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (3.5% MeOH in DCM,+0.5% Et₃N) to yield 25 (130 mg, mixture of two isomers, 69%) as a whitefoam.

Data for 25: R_(f)=0.60 (10% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.54, 9.47 (2s, 1H, NH), 8.54, 8.52 (2s, 1H,NCHN(CH₃)₂), 8.02. 8.00 (2s, 1H, H—C(8)), 7.58-7.49 (m, 2H, H-arom),7.46-7.36 (m, 4H, H-arom), 7.25 (dd, J=11.0, 3.5 Hz, 2H, H-arom),7.21-7.13 (m, 1H, H-arom), 6.80 (dd, J=8.8, 2.2 Hz, 4H, H-arom),6.00-5.82 (m, 1H, H—C(1′)), 4.16 (dd, J=10.7, 5.4 Hz, 1H, H—C(5′)),4.00-3.82 (m, 2H, H—C(4′), H—C(7′)), 3.77, 3.77 (2s, 6H, MeO), 3.62 (dt,J=12.2, 6.1 Hz, 2H, OCH₂CH₂CN), 3.51-3.33 (m, 2H, (Me₂CH)₂N), 3.15, 3.14(2s, 3H, NCHN(CH₃)₂), 3.07 (s, 3H, NCHN(CH₃)₂), 2.85-2.61 (m, 2H, C(2′),C(3′)), 2.59-2.44 (m, 2H, OCH₂CH₂CN), 2.00-1.79 (m, 2H, H—(C2′),H—C(6′)), 1.53-1.26 (m, 1H, H—C(6′)), 1.10, 1.01 (2t, J=6.4 Hz, 12H,(Me₂CH)₂N).

¹³C NMR (101 MHz, CDCl₃) δ 158.50 (MeO—C-arom), 158.04, 158.00 (C(2)),157.93 (C(6)), 156.61, 156.60 (NCHN(CH₃)₂), 149.73, 149.72 (C(4)),145.62, 145.62, 136.97, 136.94 (C-arom), 136.14 (C(8)), 130.27, 130.24,130.22, 128.26, 127.81, 126.73 (CH-arom), 120.81, 120.76 (C(5)), 117.67,117.56 (OCH₂CH₂CN), 113.10 (CH-arom), 86.88, 86.85 (C(Ph)₃), 85.58,85.37 (C(1′)), 82.41, 82.07 (C(4′)), 77.08, 76.01 (J_(C,P)=37.0, 15.1Hz, C(7′)), 74.52, 74.46 (C(5′)), 58.19, 57.74 (J_(C,P)=18.9, 19.0 HzOCH₂CH₂CN), 55.25, 55.21 (MeO-DMTr), 49.10, 48.83 (J_(C,P)=2.2, 4.8 Hz,C(3′)), 43.12, 43.00 ((Me₂CH)₂N), 41.34, 41.33 (NCHN(CH₃)₂), 38.48,38.41 (C(2′)), 37.23, 36.92 (J_(C,P)=5.7, 3.3 Hz C(6′)), 35.17((Me₂CH)₂N), 24.56, 24.53, 24.48, 24.47, 24.43, 25.36, 24.35 (7s,Me₂CH)₂N), 20.39, 20.28 (J_(C,P)=7.1, 6.9 Hz, OCH₂CH₂CN).

³¹P NMR (122 MHz, CDCl₃) δ 147.69, 146.37.

ESI⁺-HRMS m/z calcd for C₄₅H₅₆O₇N₈P ([M+H]⁺) 851.4004, found 851.4018.

Example 26(3'S,5′R,7′R)-1-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-β-D-ribofuranosyl}uracil (26)

To a solution of the sugar 6 (669 mg, 1.62 mmol) in dry DCM (13 mL) wasadded 2,6-lutidine (0.94 mL, 8.10 mmol) at 0° C. After stirring for 20min at 0° C., TMSOTf (0.89 mL, 4.86 mmol) was added dropwise and thenthe solution was allowed to warm to rt and was stirred for an additional3 h. The reaction was then quenched by addition of satd NaHCO₃ (20 mL).The organic phase was separated and aqueous phase was further extractedwith DCM (2×20 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was dissolved in dry DCM (12mL) and then Uracil (545 mg, 4.86 mmol) and BSA (1.8 mL, 7.29 mmol) wereadded at rt. After stirring for 60 min at rt, the resulting finesuspension was cool down to 0° C. and N-iodosuccinimide (578 mg, 2.52mmol) was added. After stirring for 30 min at 0° C. and for 4 h at rt,the reaction mixture was diluted with EtOAc (50 mL) and subsequentlywashed with a 10% aq solution of Na₂S₂O₃ (30 mL) and satd NaHCO₃ (30mL). Aqueous phases were combined and extracted with DCM (2×20 mL). Thecombined organic phases were dried over MgSO₄, filtered and evaporated.

The crude product was dissolved in dry toluene (15 mL) and then Bu₃SnH(0.65 mL, 2.43 mmol) and azoisobutyronitrile (AlBN, 13 mg, 0.081 mmol)were added at rt. After heating at 95° C. for 2 h, the mixture was cooldown to rt and MeOH (7 mL) and HCl (1M in water, 1.6 mL, 1.6 mmol) wereadded. The solution was further stirred for 15 min and was then dilutedwith satd NaHCO₃ (50 mL) and extracted with DCM (3×50 mL). The combinedorganic phases were dried over MgSO₄, filtered and evaporated. The crudeproduct was purified by CC (EtOAc/hexane 4:1) to yield 26 (490 mg, 61%over three steps) as a white foam.

Data for 26: R_(f)=0.15 (EtOAc/hexane 2:1);

¹H NMR (300 MHz, CDCl₃) δ 9.95 (br, 1H, H—N(3)), 7.69 (d, J=6.4 Hz, 4H,H-arom), 7.54-7.39 (m, 7H, H—C(6), H-arom), 5.98 (dd, J=9.3, 5.6 Hz, 1H,H—C(1′)), 5.71 (d, J=8.1 Hz, 1H, H—C(5)), 4.51 (dd, J=13.7, 6.3 Hz, 2H,H—C(4′), H—C(5′)), 4.14 (br, 1H, H—C(7′)), 3.25 (br, 1H, OH), 2.74 (dd,J=17.1, 8.7 Hz, 1H, H—C(3′)), 2.26-1.87 (m, 3H, H—C(2′), H—C(6′)),1.49-1.19 (m, 1H, H—C(2′)), 1.12 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 163.65 (C(4)), 150.46 (C(2)), 139.85 (C(6)),135.69, 135.66 (CH-arom), 133.71, 133.42 (C-arom), 129.98, 129.93,127.85, 127.81 (CH-arom), 102.84 (C(5)), 86.17 (C(1′)), 81.83 (C(4′)),76.94 (C(7′)), 72.45 (C(5′)), 50.09 (C(3′)), 40.93 (C(6′)), 35.83(C(2′)), 26.91 (CH₃)₃—C—Si), 19.03 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₂₇H₃₂O₅N₂NaSi ([M+Na]⁺) 515.1973, found515.1963.

Example 27(3'S,5′R,7′R)-1-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}uracil (27)

To a solution of nucleoside 26 (438 mg, 0.889 mmol) in dry pyridine (7mL) was added DMTr-Cl (1.20 g, 3.55 mmol) at rt. The solution wasstirred for 1 day at rt and then diluted with satd NaHCO₃ (30 mL) andextracted with DCM (3×40 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (1.5% MeOH in DCM, +0.5% Et₃N) to yield 27 (601 mg, 80%) as a yellowfoam.

Data for 27: R_(f)=0.48 (EtOAc/hexane 2:1);

¹H NMR (300 MHz, CDCl₃) δ 9.26 (br, 1H, H—N(3)), 7.84 (d, J=8.1 Hz, 1H,H—C(6)), 7.40-7.08 (m, 19H, H-arom), 6.69 (dd, J=8.8, 4.9 Hz, 4H,H-arom), 5.70 (dd, J=7.8, 5.8 Hz, 1H, H—C(1′)), 5.49 (dd, J=8.1, 1.5 Hz,1H, H—C(5)), 4.24-4.11 (m, 1H, H—C(5′)), 4.05-3.95 (m, 1H, H—C(4′)),3.65 (d, J=1.7 Hz, 6H, MeO), 3.62 (d, J=3.0 Hz, 1H, H—C(7′)), 2.41 (dd,J=17.2, 8.5 Hz, 1H, H—C(3′)), 2.24 (ddd, J=13.5, 10.2, 5.7 Hz, 1H,H—C(2′)), 1.39-1.24 (m, 1H, H—C(6′)), 1.04 (dd, J=13.1, 5.7 Hz, 1H,H—C(6′)), 0.89 (dt, J=13.8, 8.3 Hz, 1H, H—C(2′)), 0.81 (s, 9H,(CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 163.58 (C(4)), 158.66 (MeO—C-arom), 150.38(C(2)), 145.61 (C-arom), 139.92 (C(6)), 136.71, 136.56 (C-arom), 135.61,135.55 (CH-arom), 133.55, 133.41 (C-arom), 130.30, 129.92, 129.84,128.16, 127.90, 127.74, 127.67, 126.90, 113.19, 113.15 (CH-arom), 102.12(C(5)), 87.41 (C(Ph)₃), 86.80 (C(1′)), 82.32 (C4′)), 75.54 (C(7′)),74.41 (C(5′)), 55.23 (MeO-DMTr), 50.05 (C(3′)), 38.49 (C(6′)), 37.53(C(2′)), 26.81 (CH₃)₃—C—Si), 18.99 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₄₈H₅₀O₇N₂NaSi ([M+Na]⁺) 817.3279, found817.3286.

Example 28(3′S,5′R,7′R)-1-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (28)

To a suspension of 1,2,4-triazole (1.83 g, 26.5 mmol) in dry MeCN (70mL), at 0° C., were added POCl₃ (0.57 mL, 6.05 mmol) followed by Et₃N(4.2 mL, 30.2 mmol). The suspension was stirred for 30 min at 0° C. andthen a solution of the nucleoside 27 (601 mg, 0.756 mmol) in dry MeCN (4mL) was added at 0° C. After for 4 h of stirring at rt, the reaction wasquenched with addition satd NaHCO₃ (20 mL), MeCN removed under reducedpressure and the resulting mixture diluted with satd NaHCO₃ (30 mL) andextracted with DCM (3×60 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated.

The crude product was then dissolved in a mixture of 1,4-dioxane (18 mL)and concd NH₄OH (18 mL). After stirring for 3 h at rt, the mixture wasreduced to half of the volume in vacuo, diluted with satd NaHCO₃ (30 mL)and extracted with DCM (3×30 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (5% MeOH in DCM, +0.5% Et₃N) to yield 28 (520 mg, 87%) as a whitefoam.

Data for 28: R_(f)=0.41 (10% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 7.96 (d, J=7.4 Hz, 1H, H—C(6)), 7.45 (d, J=7.4Hz, 2H, H-arom), 7.38-7.08 (m, 17H, H-arom), 6.73 (dd, J=8.7, 4.7 Hz,4H, H-arom), 5.73 (t, J=8.6 Hz, 2H, H—C(5), H—C(1′)), 4.32-4.16 (m, 1H,H—C(5′)), 4.03 (t, J=5.6 Hz, 1H, H—C(4′)), 3.66 (d, J=0.9 Hz, 6H, MeO),3.61 (d, J=2.9 Hz, 1H, H—C(7′)), 2.50-2.33 (m, 2H, H—C(2′), H—C(3′)),1.47-1.28 (m, 1H, H—C(6′)), 1.03 (dd, J=12.9, 5.6 Hz, 1H, H—C(6′)),0.92-0.75 (m, 10H, H—C(2′), (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 165.78 (C(4)), 158.59 (MeO—C-arom), 155.94(C(2)), 145.88 (C-arom), 140.68 (C(6)), 136.93, 136.78 (C-arom), 135.59,135.53 (CH-arom), 133.60, 133.54 (C-arom), 130.31, 129.86, 129.77,128.15, 127.88, 127.71, 127.64, 126.79, 113.18, 113.14 (CH-arom), 94.53(C(5)), 87.55 (C(Ph)₃), 87.22 (C(1′)), 82.23 (C(4′)), 75.76 (C(7′)),74.68 (C(5′)), 55.21 (MeO-DMTr), 50.18 (C(3′)), 38.25 (C(6′)), 38.08(C(2′)), 26.83 (CH₃)₃—C—Si), 19.00 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₄₄H₅₂O₆N₃Si ([M+H]⁺) 794.3620, found 794.3649.

Example 29 (3'S,5′R,7′R)—N4-Benzoyl-1-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-Dideoxy-3′,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (29)

To a solution of nucleoside 28 (519 mg, 0.653 mmol) in dry DMF (15 mL)were added Et₃N (110 μL, 0.784 mmol) followed by Bz₂O (370 mg, 1633mmol) at rt and the solution was stirred overnight. Then the solutionwas quenched by careful addition of satd NaHCO₃ (60 mL) and extractedwith DCM (3×70 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC(hexane/EtOAc 2:3, +0.5% Et₃N) to yield 29 (580 mg, 99%) as a whitefoam.

Data for 29: R_(f)=0.51 (EtOAc);

¹H NMR (300 MHz, CDCl₃) δ 8.61 (d, J=7.4 Hz, 1H, H—C(6)), 7.81 (d, J=7.5Hz, 2H, H-arom), 7.49-7.13 (m, 24H, H-arom, H—C(5)), 6.77 (dd, J=8.5,4.4 Hz, 4H, H-arom), 5.73 (t, J=6.4 Hz, 1H, H—C(1′)), 4.39-4.20 (m, 1H,H—C(5′)), 4.05 (t, J=6.1 Hz, 1H, H—C(4′)), 3.70 (s, 6H, MeO), 3.63 (d,J=2.3 Hz, 1H, H—C(7′)), 2.72-2.55 (m, 1H, H—C(2′)), 2.48 (dd, J=16.0,8.4 Hz, 1H, H—C(3′)), 1.42-1.29 (m, 1H, H—C(6′)), 1.19-1.11 (m, 1H,H—C(6′)), 1.07-0.96 (m, 1H, H—C(2′)), 0.85 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 166.64 (CONH), 162.25 (C(4)), 158.70(MeO—C-arom), 154.84 (C(2)), 145.71 (C-arom), 144.84 (C(6)), 136.74,136.67 (C-arom), 135.59, 135.51 (CH-arom), 133.52, 133.42, 133.24(C-arom), 133.11, 130.30, 129.92, 129.85, 129.02, 128.12, 127.97,127.76, 127.68, 127.61, 126.94, 113.25, 113.22 (CH-arom), 96.22 (C(5)),89.07 (C(Ph)₃), 87.53 (C(1′)), 83.46 (C(4′)), 75.59 (C(7′)), 74.71(C(5′)), 55.24 (MeO-DMTr), 50.35 (C(3′)), 38.61 (C(6′)), 38.15 (C(2′)),26.82 (CH₃)₃—C—Si), 19.00 (CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₅₅H₅₆O₇N₃Si ([M+H]⁺) 898.3882, found 898.3898.

Example 30 (3′S,5′R,7′R)—N4-Benzoyl-1-{2′,3′-Dideoxy-3′,5′-ethano-7′-hydroxy-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (30)

To a solution of 29 (580 mg, 0.648 mmol) in dry THF (14 mL) was addedTBAF (1M in THF, 3.25 mL, 3.25 mmol) at rt. The solution was stirred for1 day and then was diluted with satd NaHCO₃ (50 mL) and extracted withDCM (3×40 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC (3% MeOHin DCM, +0.5% Et₃N) to yield 30 (366 mg, 85%) as a white foam.

Data for 30: R_(f)=0.31 (5% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 8.90 (br, 1H, NH), 8.73 (d, J=7.5 Hz, 1H,H—C(6)), 7.82 (d, J=7.3 Hz, 2H, H-arom), 7.55-7.31 (m, 10H, H-arom,H—C(5)), 7.28-7.09 (m, 3H, H-arom), 6.76 (dd, J=8.8, 1.7 Hz, 4H,H-arom), 5.73 (t, J=6.3 Hz, 1H, H—C(1′)), 4.28-4.13 (m, 1H, H—C(5′)),3.83 (t, J=6.0 Hz, 1H, H—C(4′)), 3.75 (d, J=3.6 Hz, 1H, H—C(7′)), 3.70(s, 6H, MeO), 2.86 (d, J=14.7 Hz, 1H, H—C-(2′)), 2.54 (dd, J=17.4, 7.4Hz, 1H, H—C(3′)), 1.68-1.55 (m, 1H, H—C(6′)), 1.45-1.13 (m, 3H, H—C(2′),H—C(6′), OH).

¹³C NMR (75 MHz, CDCl₃) δ 166.63 (CONH), 162.34 (C(4)), 158.65(MeO—C-arom), 155.00 (C(2)), 145.62 (C-arom), 145.11 (C(6)), 136.72,136.64, 133.16 (C-arom), 130.25, 129.02, 128.12, 127.93, 127.61, 126.95,113.20 (CH-arom), 96.24 (C(5)), 89.20 (C(Ph)₃), 87.48 (C(1′)), 83.40(C(4′)), 74.50, (C(5′)) 73.90 (C(7′)), 55.25 (MeO-DMTr), 50.05 (C(3′)),38.90 (C(6′)), 38.40 (C(2′)).

ESI⁺-HRMS m/z calcd for C₃₉H₃₈O₇N₃ ([M+H]⁺) 660.2704, found 660.2707.

Example 31N4-Benzoyl-1-{7′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2′,3′-Dideoxy-3′,5′-ethano-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (31)

To a solution of the nucleoside 30 (67 mg, 0.101 mmol) and5-(Ethylthio)-1H-tetrazole (22 mg, 0.17 mmol) in dry DCM (3 mL) wasadded dropwise 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(65 μL, 0.20 mmol) at rt. After stirring for 40 min, the reactionmixture was diluted with DCM (20 mL) and washed with satd NaHCO₃ (2×15mL) and satd NaCl (15 mL). Aqueous phases were combined and extractedwith DCM (20 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC (EtOAc,+0.5% Et₃N) to yield 31 (75 mg, mixture of two isomers, 86%) as a whitefoam.

Data for 31: R_(f)=0.67 (4% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 8.88 (s, 1H, NH), 8.79 (d, J=7.5 Hz, 1H,H—C(6)), 7.93 (d, J=7.5 Hz, 2H, H-arom), 7.67-7.40 (m, 10H, H-arom,H—C(5)), 7.39-7.22 (m, 3H, H-arom), 6.93-6.79 (m, 4H, H-arom), 5.97-5.77(m, 1H, H—C(1′)), 4.22 (dt, J=14.5, 5.6 Hz, 1H, H-(5′)), 3.98-3.84 (m,2H, H—C(4′), H—C(7′)), 3.82 (s, 6H, MeO), 3.66 (ddd, J=16.8, 13.5, 6.7Hz, 2H, OCH₂CH₂CN), 3.53-3.37 (m, 2H, (Me₂CH)₂N), 3.14-2.93 (m, 1H,H—C(2′)), 2.84-2.66 (m, 1H, H—C(3′)), 2.53 (dt, J=12.4, 6.3 Hz, 2H,OCH₂CH₂CN), 1.83-1.56 (m, 2H, H—C(6′)), 1.46 (td, J=14.1, 7.0 Hz, 1H,H—C(2′)), 1.18-0.97 (m, 12H, (Me₂CH)₂N).

¹³C NMR (75 MHz, CDCl₃) δ 166.70 (CONH), 162.32, 162.28 (C(4)), 158.68(MeO—C-arom), 154.93 (C(2)), 145.53 (C-arom), 144.95, 144.89 (C(6)),136.69, 136.63, 136.56, 136.52, 133.24 (C-arom), 133.10, 130.24, 130.20,129.01, 128.10, 127.94, 127.60, 126.96 (CH-arom), 117.53 (OCH₂CH₂CN),113.20 (CH-arom), 96.24 (C(5)), 89.15, 89.10 (C(Ph)₃), 87.55, 87.54(C(1′)), 83.11, 83.04 (C(4′)), 75.93, 75.37 (J_(C,P)=16.7, 15.5 Hz,C(7′)), 74.48 (C(5′)), 58.25, 57.99 (J_(C,P)=17.9, 18.1 Hz OCH₂CH₂CN),55.27, 55.24 (MeO-DMTr), 49.27, 49.03 (J_(C,P)=3.1, 4.8 Hz, C(3′)),43.15, 42.98 ((Me₂CH)₂N), 38.89, 38.80 (C(2′)), 37.44, 37.24(J_(C,P)=5.2, 3.2 Hz, C(6′)), 24.58, 24.54, 24.48, 24.45, 24.35 (5s,Me₂CH)₂N), 20.33, 20.24 (J_(C,P)=5.8, 5.7 Hz, OCH₂CH₂CN).

³¹P NMR (121 MHz, CDCl₃) δ 147.19, 146.94.

ESI⁺-HRMS m/z calcd for C₄₈H₅₅O₈N₅P ([M+H]⁺) 860.3783, found 860.3791.

Example 32(3'S,5′R,7′R)-1-{2′,3′-Dideoxy-3′,5′-ethano-7′-O-(4-nitrobenzoate)-5′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}Thymine (32)

To a solution of nucleoside 11 (100 mg, 0.175 mmol) and4-Dimethylaminopyridine (26 mg, 0.21 mmol) in dry DCM (8 mL) was added4-Nitrobenzoyl chloride (59 mg, 0.315 mmol) at rt. After stirring for 6h, the reaction is quenched by addition of satd NaHCO₃ (5 mL). Themixture is then diluted with satd NaHCO₃ (15 mL) and extracted with DCM(3×15 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (2.5% MeOH in DCM,+0.5% Et₃N) to yield 32 (98 mg, 78%) as a white foam, containing tracesof Et₃N.

Data for 32: R_(f)=0.42 (5% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 8.26 (t, J=7.3 Hz, 3H, H-arom, HN(3)), 8.00(d, J=8.9 Hz, 2H, H-arom), 7.72 (d, J=1.0 Hz, 1H, H—C(6)), 7.55 (d,J=6.9 Hz, 2H, H-arom), 7.44 (dd, J=8.8, 6.6 Hz, 4H, H-arom), 7.35-7.18(m, 3H, H-arom), 6.83 (dd, J=9.0, 2.6 Hz, 4H, H-arom), 6.01 (dd, J=8.2,5.2 Hz, 1H, H—C(1′)), 4.96 (d, J=3.3 Hz, 1H, H—C(7′)), 4.33-4.24 (m, 1H,H—C(4′)), 4.24-4.13 (m, 1H, H—C(5′)), 3.78 (d, J=0.9 Hz, 6H, MeO),2.92-2.72 (m, 2H, H—C(3′), H—C(2′)), 1.81 (d, J=0.6 Hz, 3H, Me-C(5)),1.79-1.62 (m, 2H, H—C(6′)), 1.22 (d, J=5.9 Hz, 1H, H—C(2′)).

¹³C NMR (75 MHz, CDCl₃) δ 164.05, 163.84 (C(4), CO₂R), 158.81(MeO—C-arom), 150.64, 150.52 (O₂N—C-arom, C(2)), 145.29, 136.43, 136.34(C-arom), 135.18 (C(6)), 130.62, 130.20, 130.17, 128.16, 128.01, 127.15,123.58, 113.30, 113.27 (C-arom), 111.17 (C(5)), 87.53 (C(Ph)₃), 86.29(C(1′)), 81.59 (C(4′)), 78.65 (C(7′)), 74.16 (C(5′)), 55.26 (MeO-DMTr),47.07 (C(3′)), 37.35 (C(2′)), 35.71 (C(6′)), 12.51 (Me-C(5)).

ESI⁺-HRMS m/z calcd for C₄₀H₃₇O₁₀N₃Na ([M+Na]⁺) 742.2371, found742.2375.

Example 33((3'S,5′R,7′R)-1-{2′,3′-Dideoxy-3′,5′-ethano-7′-O-(4-nitrobenzoate)-β-D-ribofuranosyl}Thymine (33)

To a solution of 32 (60 mg, 0.083 mmol) in a mixture of dry DCM (1 mL)and MeOH (0.4 mL), was added dropwise dichloroacetic acid (0.2 mL) atrt. After stirring for 3 h, the mixture is then diluted with satd NaHCO₃(15 mL) and extracted with DCM (3×10 mL). The combined organic phaseswere dried over MgSO₄, filtered and evaporated. The crude product waspurified by CC (5% MeOH in DCM) to yield 33 (29 mg, 84%) as a whitefoam. Crystals suitable for X-ray analysis were obtain byrecrystallization in a mixture of H₂O/MeOH.

Data for 33: R_(f)=0.18 (5% MeOH in DCM);

¹H NMR (400 MHz, DMSO) δ 11.33 (s, 1H, H—N(3)), 8.34 (d, J=8.8 Hz, 2H,H-arom), 8.27-8.13 (m, 2H, H-arom), 7.78 (s, 1H, H—C(6)), 5.96 (dd,J=9.3, 5.6 Hz, 1H, H—C(1′)), 5.18 (t, J=3.8 Hz, 1H, H—C(7′)), 5.12 (d,J=6.0 Hz, 1H, OH), 4.33 (dd, J=7.3, 4.7 Hz, 1H, H—C(4′)), 4.27 (td,J=10.5, 5.5 Hz, 1H, H—C(5′)), 2.90 (dd, J=17.2, 8.5 Hz, 1H, H—C(3′)),2.58-2.46 (m, 1H, H—C(2′)), 2.30 (ddd, J=13.8, 8.8, 5.3 Hz, 1H,H—C(6′)), 2.03 (dd, J=9.6, 4.2 Hz, 1H, H—C(6′)), 1.92-1.76 (m, 4H,H—C(2′), Me-C(5)).

¹³C NMR (101 MHz, DMSO) δ 164.33, 164.23 (C(4), CO₂R), 150.91, 150.75(O₂N—C-arom, C(2)), 136.79 (C-arom), 135.69 (C(6)), 131.20, 124.32(CH-arom), 109.89 (C(5)), 85.31 (C(1′)), 81.48 (C(4′)), 80.07 (C(7′)),71.72 (C(5′)), 47.18 (C(3′)), 37.77 (C(6′)), 35.48 (C(2′)), 12.66 12.58(Me-C(5)).

ESI⁺-HRMS m/z calcd for C₁₉H₂₀O₈N₃ ([M+H]⁺) 418.1245, found 418.1242.

Example 35(3′R,5′R,7′R)-1-{5′-O-acetyl-7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-dideoxy-3′,5′-ethano-α,β-D-ribofuranosyl}Thymine (35)

To a solution of the sugar 7 (933 mg, 2.05 mmol) and thymine (372 mg,3.08 mmol) in dry MeCN (12 mL) was added dropwise BSA (1.5 mL, 6.15mmol) at rt. After stirring for 50 min at rt, the solution was cooleddown to 0° C. and TMSOTf (0.45 mL, 2.5 mmol) was added dropwise. Afterfurther stirring for 3 h at 0° C. and for 15 h at rt, the reactionmixture was diluted with satd NaHCO₃ (100 mL) and extracted with DCM(4×40 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (2.5% isopropanolin DCM) to yield a mixture of 35 (924 mg, 82%) in an anomeric ratioα/β≈85:15 as a white foam.

Data for 35: R_(f)=0.56 (7% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.14 (br, 1H, H—N(3)), 7.53 (dd, J=7.7, 1.6Hz, 4H, H-arom), 7.39-7.23 (m, 6H, H-arom), 7.09 (d, J=1.0 Hz, 0.15H,H—C(6)), 6.87 (d, J=1.0 Hz, 0.85H, H—C(6)), 5.83 (t, J=6.2 Hz, 0.85H,H—C(1′)), 5.80-5.70 (m, 0.15H, H—C(1′)), 5.36-5.04 (m, 1H, H—C(5′)),4.89 (dd, J=6.3, 5.2 Hz, 1H, H—C(4′)), 4.62 (dd, J=7.1, 5.6 Hz, 0.15H,H—C(4′)), 4.01-3.85 (m, 1H, H—C(7′)), 2.76-2.55 (m, 1H, H—C(3′)),2.09-1.91 (m, 4H, H—C(6′), MeCO₂), 1.90-1.58 (m, 6H, H—C(6′), H—C(2′),Me-C(5)), 0.96 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 170.70 (MeCO₂), 163.87 (C(4)), 150.29 (C(2)),135.69, 135.67 (CH-arom), 134.99 (C(6)), 133.58, 133.18 (C-arom),130.03, 127.87 (CH-arom), 111.05 (C(5)), 87.56 (C(1′)), 82.85 (C(4′)),76.50 (C(7′)), 74.76 (C(5′)), 50.72 (C(3′)), 37.79 (C(6′)), 36.94(C(2′)), 26.88 ((CH₃)₃—C—Si), 20.95 (MeCO₂), 19.01 ((CH₃)₃—C—Si), 12.63(Me-C(5)).

ESI⁺-HRMS m/z calcd for C₃₀H₃₇O₆N₂Si ([M+H]⁺) 549.2415, found 549.2401.

Example 36(3'S,5′R,7′R)-1-{5′-O-Acetyl-2,3′-dideoxy-3,5′-ethano-7′-hydroxy-α,β-D-ribofuranosyl}thymine (36)

To a solution of the nucleoside 35 (924 mg, 1.68 mmol) in dry THF (10mL) was added TBAF (1M in THF, 3.4 mL, 3.4 mmol) at rt. After stirringfor 2 h at rt, the reaction mixture was diluted with satd NaHCO₃ (80 mL)and extracted with EtOAc (3×80 mL) and DCM (2×80 mL). The combinedorganic phases were dried over MgSO₄, filtered and evaporated. The crudeproduct was purified by CC (5% MeOH in DCM) to yield an anomeric mixtureof 36 (391 mg, 75%).

Data for 36: R_(f)=0.24 (7% MeOH in DCM);

¹H NMR (400 MHz, CDCl₃) δ 9.66 (br, 0.15H, H—N(3)), 9.63 (br, 0.85H,H—N(3)), 7.27 (d, J=1.0 Hz, 0.15H, H—C(6)), 7.06 (d, J=1.0 Hz, 0.85H,H—C(6)), 6.00 (t, J=6.1 Hz, 0.85H, H—C(1′)), 5.91 (dd, J=8.8, 5.5 Hz,0.15H, H—C(1′)), 5.26-5.10 (m, 1H, H—C(5′)), 4.92 (dd, J=6.5, 5.3 Hz,0.85H, H—C(4′)), 4.65 (dd, J=6.9, 5.7 Hz, 0.15H, H—C(4′)), 4.19-4.03 (m,1H, H—C(7′)), 2.91-2.72 (m, 2H, H—C(3′), OH), 2.64 (ddd, J=13.3, 9.8,5.5 Hz, 0.15H, H—C(2′)), 2.25-2.15 (m, 1.70H, H—C(2′)), 2.05 (s, 0.45H,MeCO₂), 2.04 (s, 2.55H, MeCO₂), 2.03-1.89 (m, 2H, H—C(6′)), 1.88 (d,J=0.7 Hz, 0.45H, Me-C(5)), 1.85 (d, J=0.6 Hz, 2.55H, Me-C(5)), 1.42-1.28(m, 0.15H, H—C(2′)).

¹³C NMR (101 MHz, CDCl₃) δ 170.87 (MeCO₂), 164.26 (C(4)), 150.66 (C(2)),135.54 (C(6)), 111.22 (C(5)), 87.97 (C(1′)), 82.97 (C(4′)), 75.08(C(7′)), 74.52 (C(5′)), 50.07 (C(3′)), 37.81 (C(2′)), 37.23 (C(6′)),21.02 (MeCO₂), 12.67 (Me-C(5)).

ESI⁺-HRMS m/z calcd for C₁₄H₁₉O₆N₂ ([M+H]⁺) 311.1238, found 311.1234.

Example 37(3'S,5′R,7′R)-1-{5′-O-Acetyl-2′,3′-dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α,β-D-ribofuranosyl}Thymine (37)

To a solution of the nucleoside 36 (364 mg, 1.17 mmol) in dry pyridine(7 mL) was added DMTr-Cl (1.19 g, 3.51 mmol) at rt. The solution wasstirred for 1 day and then was diluted with satd NaHCO₃ (50 mL) andextracted with DCM (3×50 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (EtOAc/hexane 2:1, +0.5% Et₃N) to yield an anomeric mixture of 37(690 mg, 96%) as a yellow foam.

Data for 37: R_(f)=0.70 (8% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.17 (br, 0.85H, H—N(3)), 8.56 (br, 0.15H,H—N(3)), 7.38-7.32 (m, 2H, H-arom), 7.29-7.15 (m, 7H, H-arom), 6.82 (d,J=1.1 Hz, 1H, H—C(6)), 6.76 (d, J=8.9 Hz, 4H, H-arom), 5.86 (t, J=6.0Hz, 0.85H, H—C(1′)), 5.71 (dd, J=8.9, 5.4 Hz, 0.15H, H—C(1′)), 5.25 (dd,J=10.2, 5.6 Hz, 0.15H, H—C(5′)), 5.21-5.11 (m, 0.85H, H—(C5′)), 4.78(dd, J=6.7, 4.8 Hz, 0.85H, H—C(4′)), 4.49 (dd, J=7.1, 5.3 Hz, 0.15H,H—C(4′)), 3.84 (br, 1H, H—C(7′)), 3.72, 3.71 (2s, 6H, MeO), 2.34-2.23(m, 1H, H—C(3′)), 2.01, 1.99 (2s, 3H, MeCO₂), 1.82 (d, J=0.5 Hz,Me-C(5)), 1.80-1.56 (m, 4H, H—C(2′), H—C(6′)).

¹³C NMR (75 MHz, CDCl₃) δ 170.69 (MeCO₂), 163.91 (C(4)), 158.82(MeO—C-arom), 150.33 (C(2)), 145.34, 136.64, 136.58 (C-arom), 135.00(C(6)), 130.25, 128.39, 128.07, 127.15, 113.41 (CH-arom), 111.04 (C(5)),87.70 (C(Ph)₃), 87.31 (C(1′)), 83.15 (C(4′)), 77.16 (C(7′)), 74.96(C(5′)), 55.37 (MeO-DMTr), 49.12 (C(3′)), 37.55 (C(2′)), 36.82 (C(6′)),21.07 (MeCO₂), 12.66 (Me-C(5)).

ESI⁺-HRMS m/z calcd for C₃₅H₃₆O₈N₂ ([M+H]⁺) 612.2466, found 612.2453.

Example 38(3'S,5′R,7′R)-1-{2′,3′-Dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}thymine (38)

To a solution of the nucleoside 37 (690 mg, 1.12 mmol) in dry MeOH (10mL) was added K₂CO₃ (467 mg, 3.36 mmol) at rt. The solution was stirredfor 3 h and then diluted with satd NaCl (60 mL) and extracted with DCM(3×60 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (3% isopropanol inEt₂O, +0.5% Et₃N) to yield the α-anomer 38 (550 mg, 86%) as a whitesolid.

Data for 38: R_(f)=0.39 (5% MeOH in DCM);

¹H NMR (400 MHz, CDCl₃) δ 9.37 (br, s, 1H, H—N(3)), 7.39-7.31 (m, 2H,H-arom), 7.25 (d, J=8.3 Hz, 4H, H-arom), 7.20 (t, J=7.7 Hz, 2H, H-arom),7.16-7.08 (m, 1H, H-arom), 6.78 (d, J=1.1 Hz, 1H, H—C(6)), 6.74 (d,J=8.8 Hz, 4H, H-arom), 5.91 (dd, J=6.5, 4.9 Hz, 1H, H—C(1′)), 4.57 (dd,J=7.2, 4.4 Hz, 1H, H—C(4′)), 4.35-4.18 (m, 1H, H—C(5′)), 3.86 (d, J=4.7Hz, 1H, H—C(7′)), 3.69 (s, 6H, MeO), 2.53 (br, 1H, OH), 2.22 (dd,J=15.3, 6.3 Hz, 1H, H—C(3′)), 1.85-1.69 (m, 5H, Me-C(5), H—C(2′),H—C(6′)), 1.66-1.49 (m, 2H, H—C(2′), H—C(6′)).

¹³C NMR (101 MHz, CDCl₃) δ 163.98 (C(4)), 158.67 (MeO—C-arom), 150.47(C(2)), 145.48, 136.80, 136.75 (C-arom), 134.94 (C(6)), 130.19, 130.18,128.35, 127.97, 127.01, 113.31 (CH-arom), 111.04 (C(5)), 87.82 (C(Ph)₃),87.05 (C(1′)), 85.74 (C(4′)), 78.26 (C(7′)), 73.33 (C(5′)), 55.31(MeO-DMTr), 48.81 (C(3′)), 40.21 (C(6′)), 37.68 (C(2′)), 12.65(Me-C(5)).

ESI⁺-HRMS m/z calcd for C₃₃H₃₅O₇N₂ ([M+H]⁺) 571.2439, found 571.2421.

Example 39(3'S,5′R,7′R)-1-{5′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]2′,3′-Dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}Thymine (39)

To a solution of the nucleoside 38 (200 mg, 0.350 mmol) and5-(Ethylthio)-1H-tetrazole (59 mg, 0.46 mmol) in dry DCM (7 mL) wasadded dropwise 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(0.17 mL, 0.53 mmol) at rt. After stirring for 1 h, the reaction mixturewas diluted with DCM (50 mL) and washed with satd NaHCO₃ (2×25 mL) andsatd NaCl (25 mL). Aqueous phases were combined and extracted with DCM(30 mL). The combined organic phases were dried over MgSO₄, filtered andevaporated. The crude product was purified by CC (2% MeOH in DCM, +0.5%Et₃N) to yield 39 (220 mg, mixture of two isomers, 81%) as a whitesolid.

Data for 39: R_(f)=0.44 (4% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.03 (br, 1H, H—N(3)), 7.36 (d, J=8.1 Hz, 2H,H-arom), 7.30-7.07 (m, 7H, H-arom), 6.84 (s, 1H, H—C(6)), 6.80-6.69 (m,4H, H-arom), 5.95, 5.88 (2dd, J=6.6, 4.8 Hz, 1H, H—C(1′)), 4.70, 4.61(2dd, J=7.3, 4.3 Hz, 1H, H—C(4′)), 4.41-4.20 (m, 1H, H—C(5′)), 3.94-3.82(m, 1H, H—C(7′)), 3.81-3.62 (m, 8H, MeO, OCH₂CH₂CN), 3.59-3.40 (m, 2H,(Me₂CH)₂N), 2.61-2.46 (m, 2H, OCH₂CH₂CN), 2.28 (ddd, J=14.1, 13.2, 7.3Hz, 1H, H—C(3′)), 1.91-1.73 (m, 5H, Me-C(5), H—C(6′), H—C(2′)),1.72-1.46 (m, 2H, H—C(6′), H—C(2′)), 1.16-1.00 (m, 12H, (Me₂CH)₂N).

¹³C NMR (75 MHz, CDCl₃) δ 164.01, 163.98 (C(4)), 158.70 (MeO—C-arom),150.39, 150.17 (C(2)), 145.52, 136.84, 136.78 (C-arom), 135.44, 135.39(C(6)), 130.21, 128.36, 128.32, 128.00, 127.03 (CH-arom), 118.02, 117.76(OCH₂CH₂CN), 113.32 (CH-arom), 110.91, 110.59 (C(5)), 88.31, 88.06(C(Ph)₃), 87.11, 87.06 (C(1′)), 85.44, 85.39 (J_(C,P)=4.6, 3.1 Hz,C(4′)), 78.25, 78.13 (C(7′)), 74.70, 74.34 (J_(C,P)=13.5, 18.5 Hz,C(5′)), 58.73, 58.47 (J_(C,P)=18.9, 20.1 Hz, (OCH₂CH₂CN)), 55.35, 55.32(MeO-DMTr), 48.80, 48.64 (C(3′)), 43.22, 43.06 (J_(C,P)=12.4, 11.0 Hz(Me₂CH)₂N), 39.68, 39.63 (C(6′)), 38.06, 37.93 (C(2′)), 24.81, 24.74,24.71, 24.68, 24.65, 24.59 (6s, Me₂CH)₂N), 20.37, 20.35 (J_(C,P)=7.1,6.8 Hz, OCH₂CH₂CN), 12.66 (Me-C(5)).

³¹P NMR (122 MHz, CDCl₃) δ 148.18, 147.80.

ESI⁺-HRMS m/z calcd for C₄₂H₅₂O₈N₄P ([M+H]⁺) 771.3517, found 771.3517.

Example 40(3'S,5′R,7′R)—N4-Benzoyl-1-{2′,3′-dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}-5-methylcytosine(40)

To a solution of the nucleoside 38 (268 mg, 0.470 mmol) in dry MeCN (5mL) was added dropwise BSA (0.28 mL, 1.13 mmol) at 0°, and then thesolution was stirred overnight at rt. In another flask, a suspension of1,2,4-triazole (1.14 g, 16.5 mmol) in dry MeCN (50 mL) was cool down to0° C. and POCl₃ (0.35 mL, 3.8 mmol) followed Et₃N (2.62 mL, 18.8 mmol)were added. The suspension was stirred for 30 min at 0° C., and then theprevious prepared solution of the silylated compound 38 was added to thesuspension and the mixture was further stirred for 7 h at rt. Reactionwas quenched with addition satd NaHCO₃ (10 mL), MeCN removed underreduced pressure and the resulting mixture diluted with satd NaHCO₃ (30mL) and extracted with DCM (3×30 mL). The combined organic phases weredried over MgSO₄, filtered and evaporated.

The crude product was then dissolved in a mixture of 1,4-dioxane (10 mL)and concd NH₄OH (10 mL). After stirring for 3 h at rt, the mixture wasreduced to half of its volume in vacuo, diluted with satd NaHCO₃ (25 mL)and extracted with DCM (4×30 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated.

The crude product was then dissolved in dry DMF (10 mL). Et₃N (80 μL,0.56 mmol) followed by Bz₂O (266 mg, 1.18 mmol) were added at rt and thesolution was stirred overnight. The resulting brownish solution wasquenched by careful addition of satd NaHCO₃ (40 mL) and extracted withDCM (4×40 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC(EtOAc/hexane 1:1, +0.5% Et₃N) to yield 40 (263 mg, 83%) as a whitefoam.

Data for 40: R_(f)=0.53 (EtOAc/hexane 3:1);

¹H NMR (300 MHz, CDCl₃) δ 13.11 (br, 1H, NH), 8.30-8.10 (m, 2H, H-arom),7.47-7.29 (m, 5H, H-arom), 7.28-7.06 (m, 7H, H-arom), 7.00 (d, J=0.8 Hz,1H, H—C(6)), 6.74 (d, J=8.6 Hz, 4H, H-arom), 5.89 (dd, J=6.3, 4.6 Hz,1H, H—C(1′)), 4.61 (dd, J=7.2, 4.5 Hz, 1H, H—C(4′)), 4.33-4.20 (m, 1H,H—C(5′)), 3.87 (br, 1H, H—C(7′)), 3.69 (s, 6H, MeO), 2.32-2.13 (m, 2H,H—C(3′), OH), 1.99 (s, 3H, Me-C(5)), 1.87-1.73 (m, 2H, H—C(2′),H—C(6′)), 1.66-1.47 (m, 2H, H—C(2′), H—C(6′)).

¹³C NMR (75 MHz, CDCl₃) δ 179.61 (CONH), 159.76 (C(4)), 158.74(MeO—C-arom), 147.87 (C(2)), 145.47 (C-arom), 137.17 (C(6)), 136.77,136.68, 136.03 (C-arom), 132.55, 130.21, 129.98, 128.34, 128.21, 128.03,127.07, 113.35 (CH-arom), 111.81 (C(5)), 88.74 (C(Ph)₃), 87.13 (C(1′)),86.12 (C(4′)), 78.17 (C(7′)), 73.31 (C(5′)), 55.35 (MeO-DMTr), 48.63(C(3′)), 40.35 (C(6′)), 38.06 (C(2′)), 13.78 (Me-C(5)).

ESI⁺-HRMS m/z calcd for C₄₀H₄₀O₇N₃ ([M+H]⁺) 674.2861, found 674.2877.

Example 41(3'S,5′R,7′R)—N4-Benzoyl-1-{5′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]2′,3′-dideoxy-3,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}-5-methylcytosine(41)

To a solution of the nucleoside 40 (250 mg, 0.371 mmol) and5-(Ethylthio)-1H-tetrazole (73 mg, 0.56 mmol) in dry DCM (8 mL) wasadded dropwise 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(0.20 mL, 0.63 mmol) at rt. After stirring for 30 min, the reactionmixture was diluted with DCM (30 mL) and washed with satd NaHCO₃ (2×20mL) and satd NaCl (20 mL). Aqueous phases were combined and extractedwith DCM (20 mL).

The combined organic phases were dried over MgSO₄, filtered andevaporated. The crude product was purified by CC (EtOAc/hexane 1:1,+0.5% Et₃N) to yield 41 (260 mg, mixture of two isomers, 80%) as a whitefoam.

Data for 41: R_(f)=0.57 (EtOAc/hexane 1:1);

¹H NMR (300 MHz, CDCl₃) δ 13.26 (br, 1H, NH), 8.32 (d, J=7.2 Hz, 2H,H-arom), 7.58-7.39 (m, 5H, H-arom), 7.38-7.14 (m, 8H, H-arom, H—C(6)),6.88-6.77 (m, 4H, H-arom), 6.01, 5.96 (2dd, J=6.3, 4.6 Hz, 1H, H—C(1′)),4.82, 4.74 (2dd, J=7.3, 4.3 Hz, 1H, H—C(4′)), 4.42 (td, J=10.6, 6.0 Hz,1H, H—C(5′)), 3.97 (br, 1H, H—C(7′)), 3.91-3.68 (m, 8H, MeO, OCH₂CH₂CN),3.59 (dtd, J=16.7, 6.7, 3.4 Hz, 2H, (Me₂CH)₂N)), 2.62 (dt, J=15.5, 6.4Hz, 2H, OCH₂CH₂CN), 2.49-2.23 (m, 1H, H—C(3′)), 2.11, 2.09 (2d, J=0.5Hz, 3H, Me-C(5)), 2.00-1.82 (m, 2H, H—C(6′), H—C(2′)), 1.82-1.55 (m, 2H,H—C(6′), H—C(2′)), 1.17 (dd, J=16.3, 6.8 Hz, 12H, (Me₂CH)₂N).

¹³C NMR (101 MHz, CDCl₃) δ 179.60 (CONH), 159.97 (C(4)), 158.76(MeO—C-arom), 147.81, 147.70 (C(2)), 145.54 (C-arom), 137.34, 136.83(C(6)), 136.77, 136.72, 136.65, 136.55 (C-arom), 132.45, 130.22, 130.20,129.96, 128.34, 128.31, 128.18, 128.00, 127.04 (CH-arom), 117.89, 117.71(OCH₂CH₂CN), 113.35 (CH-arom), 111.60, 111.36 (C(5)), 89.24, 89.01(C(Ph)₃), 87.16, 87.12 (C(1′)), 85.78, 85.62 (J_(C,P)=4.3, 3.2 Hz,C(4′)), 78.20, 77.98 (C(7′)), 74.68, 74.37 (J_(C,P)=13.4, 18.2 Hz,C(5′)), 58.70, 58.44 (J_(C,P)=18.5, 20.0 Hz, (OCH₂CH₂CN)), 55.36, 55.33(MeO-DMTr), 48.65, 48.44 (C(3′)), 43.27, 43.14 (J_(C,P)=12.4, 12.3 Hz(Me₂CH)₂N), 39.87, 39.64 (J_(C,P)=3.4, 3.7 Hz (C(6′)), 38.30, 38.22(C(2′)), 24.80, 24.72, 24.70, 24.67, 24.63 (Me₂CH)₂N), 20.39, 20.37(J_(C,P)=7.2, 6.8 Hz, OCH₂CH₂CN), 13.72 (Me-C(5)).

³¹P NMR (121 MHz, CDCl₃) δ 148.18, 147.96.

ESI⁺-HRMS m/z calcd for C₄₉H₅₇O₈N₅P ([M+H]⁺) 874.3939, found 874.3946.

Example 42(3′R,5′R,7′R)—N6-Benzoyl-9-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-dideoxy-3′,5′-ethano-α-D-ribofuranosyl}adenine (42)

The nucleoside 15 (1.74 g, 2.64 mmol) was dissolved in 0.15 M NaOH inTHF/methanol/H₂O (5:4:1, 80 mL) at 0° C. The reaction was stirred for 20min and quenched by addition of NH₄Cl (1.06 g). Solvents were thenremoved under reduced pressure and the product purified by CC (5%isopropanol in DCM) to yield 42 (α-anomer, 836 mg, 51%) and 16(β-anomer, 287 mg, 18%) as white foams.

Data for 42: R_(f)=0.35 (5% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.34 (s, 1H, NH), 8.71 (s, 1H, H—C(2)), 8.02(d, J=7.4 Hz, 2H, H-arom)), 7.92 (s, 1H, H—C(8)), 7.68-7.58 (m, 4H,H-arom), 7.58-7.31 (m, 9H, H-arom), 6.23 (dd, J=6.7, 2.4 Hz, 1H,H—C(1′)), 4.74 (dd, J=6.6, 4.9 Hz, 1H, H—C(4′)), 4.49 (dt, J=12.5, 6.3Hz, 1H, H—C(5′)), 4.10 (br, 1H, H—C(7′)), 3.07 (d, J=6.7 Hz, 1H, OH),2.92 (dd, J=15.4, 7.3 Hz, 1H, H—C(3′)), 2.52-2.35 (m, 1H, H—C(2′)),2.10-1.97 (m, 1H, H—C(6′)), 1.94-1.77 (m, 2H, H—C(2′), H—C(6′)), 1.06(s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 164.98 (CONH), 152.65 (C(2)), 151.31 (C(4)),149.69 (C(6)), 140.93 (C(8)), 135.74 (CH-arom), 133.82, 133.68, 133.39(C-arom), 132.77, 130.02, 129.98, 128.76, 128.06, 127.87, 127.85(CH-arom), 123.38 (C(5)), 87.16 (C(1′)), 85.35 (C(4′)), 77.40 (C(7′)),72.79 (C(5′)), 50.63 (C(3′)), 40.86 (C(6′)), 37.25 (C(2′)), 26.94((CH₃)₃—C—Si), 19.05 ((CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₃₅H₃₈O₄N₅Si ([M+H]⁺) 620.2688, found 620.2671.

Example 43(3′R,5′R,7′R)—N6-Benzoyl-9-{5′-O-acetyl-7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-dideoxy-3′,5′-ethano-α-D-ribofuranosyl}adenine (43)

To a solution of the nucleoside 42 (1.09 g, 1.75 mmol) and4-dimethylaminopyridine (321 mg, 2.63 mmol) in dry DCM (50 mL) was addedacetic anhydride (0.83 mL, 8.8 mmol) at rt. After stirring overnight,the reaction was quenched by addition of satd NaHCO₃ (50 mL). The phaseswere separated and aqueous phase further extracted with DCM (2×80 mL).The combined organic phases were dried over MgSO₄, filtered andevaporated. The crude product was purified by CC (2.5% MeOH in DCM) toyield 43 (1.04 g, 90%) as white foams.

Data for 43: R_(f)=0.33 (EtOAc/hexane 4:1);

¹H NMR (300 MHz, CDCl₃) δ 8.99 (br, 1H, NH), 8.73 (s, 1H, H—C(2)),8.09-7.99 (m, 2H, H-arom), 7.98 (s, 1H, H—C(8)), 7.70-7.58 (m, 5H,H-arom), 7.57-7.48 (m, 2H, H-arom), 7.47-7.34 (m, 6H, H-arom), 6.22 (dd,J=6.8, 3.2 Hz, 1H, H—C(1′)), 5.45-5.35 (m, 1H, H—C(5′)), 5.01 (dd,J=6.7, 5.0 Hz, 1H, H—C(4′)), 4.09 (d, J=4.1 Hz, 1H, H—C(7′)), 3.02 (dt,J=9.5, 6.5 Hz, 1H, H—C(3′)), 2.55 (ddd, J=13.5, 10.0, 3.2 Hz, 1H,H—C(2′)), 2.15 (dd, J=13.2, 6.2 Hz, 1H, H—C(6′)), 2.09 (s, 3H, MeCO₂),2.01 (dt, J=8.0, 3.5 Hz, 1H, H—C(2′)), 1.88 (dt, J=13.6, 5.3 Hz, 1H,H—C(6′)), 1.08 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (101 MHz, CDCl₃) δ 170.61 (MeCO₂), 164.75 (CONH), 152.67 (C(2)),151.37 (C(4)), 149.64 (C(6)), 141.41 (C(8)), 135.85 (CH-arom), 133.71,133.38 (C-arom), 132.91, 130.15, 130.10, 128.99, 128.02, 127.99, 127.97(CH-arom), 123.64 (C(5)), 87.37 (C(1′)), 83.37 (C(4′)), 76.63 (C(7′)),74.51 (C(5′)), 51.19 (C(3′)), 37.44 (C(2′)), 37.32 (C(6′)), 27.01((CH₃)₃—C—Si), 21.08 (MeCO₂), 19.14 ((CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₃₇H₄₀O₅N₅Si ([M+H]⁺) 662.2793, found 662.2787.

Example 44(3′S,5′R,7′R)—N6-Benzoyl-9-{5′-O-acetyl-2′,3′-dideoxy-3′,5′-ethano-7′-hydroxy-α-D-ribofuranosyl}adenine (44)

To a solution of the nucleoside 43 (990 mg, 1.50 mmol) in dry THF (50mL) was added TBAF (1M in THF, 3.0 mL, 3.0 mmol) at rt. After stirringfor 3.5 hours at rt, the solution was diluted with EtOAc (30 mL) and THFwas removed under reduced pressure. The mixture was then diluted withsatd NaHCO₃ (50 mL) and extracted with DCM (4×50 mL). The combinedorganic phases were dried over MgSO₄, filtered and evaporated. The crudeproduct was purified by CC (6% MeOH in DCM) to yield 44 (570 mg, 90%) asa white foam, containing traces of TBAF.

Data for 44: R_(f)=0.33 (10% MeOH in DCM);

¹H NMR (400 MHz, CDCl₃) δ 9.60 (br, 1H, NH), 8.67 (s, 1H, H—C(2)), 8.09(s, 1H, H—C(8)), 7.96 (d, J=7.4 Hz, 2H, H-arom), 7.51 (t, J=7.4 Hz, 1H,H-arom), 7.42 (t, J=7.5 Hz, 2H, H-arom), 6.33 (dd, J=6.7, 3.1 Hz, 1H,H—C(1′)), 5.25 (ddd, J=9.7, 6.4, 5.3 Hz, 1H, H—C(5′)), 4.92 (dd, J=6.4,5.4 Hz, 1H, H—C(1′)), 4.14 (br, 2H, H—C(7′), OH), 3.06 (dd, J=16.0, 6.6Hz, 1H, H—C(3′)), 2.87 (ddd, J=13.2, 9.9, 3.0 Hz, 1H, H—C(2′)),2.26-2.17 (m, 1H, H—C2′)), 2.10-1.98 (m, 5H, H—C(6′), MeCO₂).

¹³C NMR (75 MHz, CDCl₃) δ 170.64 (MeCO₂), 165.27 (CONH), 152.49 (C(2)),151.26 (C(4)), 149.58 (C(6)), 141.64 (C(8)), 133.60 (C-arom), 132.82,128.76, 128.06 (CH-arom), 123.30 (C(5)), 87.30 (C(1′)), 83.17 (C(4′)),74.67 (C(7′)), 74.20 (C(5′)), 50.41 (C(3′)), 37.43 (C(2′)), 36.92(C(6′)), 20.96 (MeCO₂).

ESI⁺-HRMS m/z calcd for C₂₁H₂₂O₅N₅ ([M+H]⁺) 424.1615, found 424.1623.

Example 45(3′S,5′R,7′R)—N6-Benzoyl-9-{5′-O-acetyl-2′,3′-dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}Adenine (45)

To a solution of nucleoside 44 (570 mg, 1.35 mmol) in dry pyridine (16mL) was added DMTr-Cl (1.37 g, 4.04 mmol) at rt. The solution wasstirred for 1 day and then was diluted with satd NaHCO₃ (100 mL) andextracted with DCM (3×80 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (2% MeOH in DCM, +0.5% Et₃N) to yield 45 (876 mg, 89%) as a yellowfoam.

Data for 45: R_(f)=0.81 (5% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.42 (d, J=14.6 Hz, 1H, NH), 8.73 (s, 1H,H—C(2)), 8.03 (d, J=7.6 Hz, 2H, H-arom), 7.93 (s, 1H, H—C(8)), 7.66-7.55(m, 1H, H-arom), 7.55-7.45 (m, 4H, H-arom), 7.45-7.22 (m, 7H, H-arom),6.87 (d, J=8.7 Hz, 4H, H-arom), 6.25 (dd, J=6.6, 2.4 Hz, 1H, H—C(1′)),5.47-5.33 (m, 1H, H—C(5′)), 4.89 (dd, J=6.7, 4.9 Hz, 1H, H—C(4′)), 4.02(d, J=2.5 Hz, 1H, H—C(7′)), 3.79 (s, 6H, MeO), 2.58 (dd, J=16.0, 6.9 Hz,1H, H—C(3′)), 2.38 (ddd, J=12.7, 10.0, 2.4 Hz, 1H, H—C(2′)), 2.11 (s,3H, MeCO₂), 2.09-1.87 (m, 3H, H—C(2′), H—C(6′)). ¹³C NMR (75 MHz, CDCl₃)δ 170.40 (MeCO₂), 164.84 (CONH), 158.66 (MeO—C-arom), 152.45 (C(2)),151.22 (C(4)), 149.51 (C(6)), 145.23 (C-arom), 141.23 (C(8)), 136.51,133.65 (C-arom), 132.68, 130.12, 128.75, 128.33, 127.95, 127.90, 127.03(CH-arom), 123.55 (C(5)), 113.27 (CH-arom), 87.19 (C(Ph)₃), 87.12(C(1′)), 83.25 (C(4′)), 77.16 (C(7′)), 74.41 (C(5′)), 55.23 (MeO-DMTr),49.23 (C(3′)), 37.61 (C(2′)), 36.22 (C(6′)), 20.98 (MeCO₂).

ESI⁺-HRMS m/z calcd for C₄₂H₄₀O₇N₅ ([M+H]⁺) 726.2922, found 726.2905.

Example 46(3'S,5′R,7′R)—N6-benzoyl-9-{2′,3′-dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}Adenine (46)

The nucleoside 45 (870 mg, 1.20 mmol) was dissolved in 0.1 M NaOH inTHF/methanol/H₂O (5:4:1, 50 mL) at 0° C. The reaction was stirred for 30min at 0° C. and then quenched by addition of NH₄Cl (321 mg). Thesolution was diluted with satd NaHCO₃ (100 mL) and extracted with DCM(4×80 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (3% MeOH in DCM,+0.5% Et₃N) to yield 46 (777 mg, 94%) as white foams.

Data for 46: R_(f)=0.26 (5% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.39 (s, 1H, NH), 8.61 (s, 1H, H—C(2)), 7.93(d, J=7.4 Hz, 2H, H-arom), 7.75 (s, 1H, H—C(8)), 7.46 (t, J=7.3 Hz, 1H,H-arom), 7.40-7.31 (m, 4H, H-arom), 7.29-7.16 (m, 6H, H-arom), 7.11 (t,J=7.2 Hz, 1H, H-arom), 6.73 (d, J=8.7 Hz, 4H, H-arom), 6.12 (dd, J=6.5,1.9 Hz, 1H, H—C(1′)), 4.53 (dd, J=7.5, 4.5 Hz, 1H, H—C(4′)), 4.32 (br,1H, H—C(5′)), 3.90 (t, J=4.5 Hz, 1H, H—C(7′)), 3.66, 3.65 (2s, 6H, MeO),3.31 (br, 1H, OH), 2.36 (dd, J=16.5, 8.1 Hz, 1H, H—C(3′)), 2.04 (ddd,J=12.0, 9.9, 2.0 Hz, 1H, H—C(2′)), 1.92-1.69 (m, 3H, H—C(2′), H—C(6′)).

¹³C NMR (75 MHz, CDCl₃) δ 164.92 (CONH), 158.64 (MeO—C-arom), 152.60(C(2)), 151.28 (C(4)), 149.61 (C(6)), 145.44 (C-arom), 140.71 (C(8)),136.77, 133.65 (C-arom), 132.72, 130.15, 130.12, 128.73, 128.39, 128.04,127.96, 127.02 (CH-arom), 123.27 (C(5)), 113.28 (CH-arom), 87.11(C(1′)), 87.01 (C(Ph)₃), 85.60 (C(4′)), 78.16 (C(7′)), 72.72 (C(5′)),55.28 (MeO-DMTr), 48.89 (C(3′)), 39.93 (C(6′)), 37.55 (C(2′)).

ESI⁺-HRMS m/z calcd for C₄₀H₃₈O₆N₅ ([M+H]⁺) 684.2817, found 684.2800.

Example 47(3′S,5′R,7′R)—N6-Benzoyl-9-{5′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2′,3′-dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}Adenine (47)

To a solution of the nucleoside 46 (199 mg, 0.290 mmol) and5-(Ethylthio)-1H-tetrazole (57 mg, 0.44 mmol) in dry DCM (7 mL) wasadded dropwise 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(0.16 mL, 0.49 mmol) at rt. After stirring for 60 min, the reactionmixture was diluted with satd NaHCO₃ (20 mL) and extracted with DCM(3×20 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (EtOAc, +0.5% Et₃N)to yield 47 (197 mg, mixture of two isomers, 77%) as a white foam.

Data for 47: R_(f)=0.75 (5% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 8.98 (br, 1H, NH), 8.68, 8.67 (2s, 1H, C(2)),7.94 (d, J=7.6 Hz, 2H, H-arom), 7.90, 7.84 (2s, 1H, C(8)), 7.56-7.49 (m,1H, H-arom), 7.48-7.34 (m, 4H, H-arom), 7.30-7.10 (m, 7H, H-arom),6.80-6.69 (m, 4H, Harom), 6.21, 6.15 (2dd, J=6.8, 2.2 Hz, 1H, H—C(1′)),4.69, 4.59 (2dd, J=7.3, 4.5 Hz, 1H, H—C(4′)), 4.44 (tt, J=12.3, 6.3 Hz,1H, H—C(5′)), 3.90 (dd, J=9.0, 3.8 Hz, 1H, H—C(5′)), 3.82-3.63 (m, 8H,MeO, OCH₂CH₂CN), 3.59-3.43 (m, 2H, (Me₂CH)₂N), 2.61-2.49 (m, 2H,OCH₂CH₂CN), 2.47-2.07 (m, 2H, H—C(3′), H—C(2′)), 1.98-1.66 (m, 3H,H—C(2′), H—C(6′)), 1.15-1.03 (m, 12H, (Me₂CH)₂N).

¹³C NMR (101 MHz, CDCl₃) δ 164.67 (CONH), 158.77 (MeO—C-arom), 152.58(C(2)), 151.34, 151.29 (C(4)), 149.46 (C(6)), 145.55, 145.54 (C-arom),141.58, 141.50 (C(8)), 136.87, 136.85, 136.84, 133.85 (C-arom), 132.85,130.26, 130.23, 130.20, 128.97, 128.47, 128.43, 128.02, 127.96, 127.08(CH-arom), 123.62, 123.58 (C(5)), 117.91, 117.70 (OCH₂CH₂CN), 113.37(CH-arom), 87.80, 87.67 (C(1′)), 87.20, 87.14 (C(Ph)₃), 85.29, 85.22((J_(C,P)=4.2, 3.1 Hz, C(4′)), 78.16, 77.96 (C(7′)), 74.28, 73.98(J_(C,P)=14.8, 18.4 Hz, C(5′)), 58.80, 58.61 (J_(C,P)=16.2, 17.3 HzOCH₂CH₂CN), 55.37, 55.35 (MeO-DMTr), 49.02, 48.91 (C(3′)), 43.29, 43.16(J_(C,P)=8.9, 9.0 Hz, ((Me₂CH)₂N), 39.09 (C(6′)), 37.99, 37.95 (C(2′)),24.82, 24.77, 24.74, 24.70, 24.64 ((Me₂CH)₂N), 20.43, 20.42(J_(C,P)=1.4, 1.9 Hz, OCH₂CH₂CN).

³¹P NMR (121 MHz, CDCl₃) δ 148.14, 148.11.

ESI⁺-HRMS m/z calcd for C₄₅H₅₆O₇N₈P ([M+H]⁺) 884.3895, found 884.3904.

Example 48(3′R,5′R,7′R)-2-Amino-6-chloro-9-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-dideoxy-3,5′-ethano-α-D-ribofuranosyl}purine (48)

The nucleoside 20 (1.78 g, 3.01 mmol) was dissolved in 0.5 M NaOH inTHF/methanol/H₂O (5:4:1, 15 mL) at 0° C. The reaction was stirred for 20min at 0° C. and was quenched by addition of NH₄Cl (484 mg). Thesuspension was then diluted with satd NaHCO₃ (100 mL) and extracted withDCM (4×75 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC (3% MeOHin DCM) to yield 48 (α-anomer, 992 mg, 60%) and 21 (β-anomer, 428 mg,25%) as white foams.

Data for 48: R_(f)=0.34 (5% MeOH in DCM);

¹H NMR (400 MHz, CDCl₃) δ 7.71-7.60 (m, 5H, H-arom, H—(C(8)), 7.49-7.34(m, 6H, H-arom), 6.08 (dd, J=6.9, 2.6 Hz, 1H, H—C(1′)), 5.26 (s, 2H,NH₂), 4.70 (dd, J=7.5, 4.8 Hz, 1H, H—C(4′)), 4.47 (dt, J=10.0, 5.1 Hz,1H, H—C(5′)), 4.11 (t, J=3.3 Hz, 1H, H—C(7′)), 2.87 (dd, J=16.5, 7.7 Hz,1H, H—C(3′)), 2.57 (br, 1H, OH), 2.27 (ddd, J=14.0, 9.9, 2.6 Hz, 1H,H—C(2′)), 2.10-2.01 (m, 1H, H—C(6′)), 1.92-1.76 (m, 2H, H—C(2′),H—C(6′)), 1.06 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 159.09 (C(2)), 153.05 (C(4)), 151.46 (C(6)),139.91 (C(8)), 135.71 (CH-arom), 133.96, 133.27 (C-arom), 130.00,129.96, 127.86, 127.83 (CH-arom), 125.52 (C(5)), 86.46 (C(1′)), 84.92(C(4′)), 77.40 (C(7′)), 72.63 (C(5′)), 50.55 (C(3′)), 40.92 (C(6′)),36.78 (C(2′)), 26.88 ((CH₃)₃—C—Si), 19.01 ((CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₂₈H₃₃O₃N₅ClSi ([M+H]⁺) 550.2036, found550.2019.

Example 49(3′R,5′R,7′R)-9-{7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-dideoxy-3′,5′-ethano-α-D-ribofuranosyl}guanine (49)

To a solution of the nucleoside 48 (610 mg, 1.03 mmol) in dry DCM (15mL) were added 3-hydroxypropionitrile (0.28 mL, 4.12 mmol) followed by1,5,7-Triazabicyclo[4.4.0]dec-5-ene (287 mg, 2.06 mmol) at rt. After 4hours of stirring at rt, a second portion of 3-hydroxypropionitrile(0.28 mL, 3.23 mmol) followed by 1,5,7-Triazabicyclo[4.4.0]dec-5-ene(287 mg, 2.06 mmol) were added. The reaction was further stirred for 2days and then was directly purified by CC (10% MeOH in DCM) to yield 49(500 mg, 87%) as white foam.

Data for 49: R_(f)=0.30 (10% MeOH in DCM);

¹H NMR (400 MHz, MeOD) δ 7.73-7.61 (m, 5H, H-arom, H—C(8)), 7.53-7.32(m, 6H, H-arom), 6.06 (dd, J=6.9, 3.7 Hz, 1H, H—C(1′)), 4.74 (dd, J=7.0,4.6 Hz, 1H, H—C(4′)), 4.46-4.36 (m, 1H, H—C(5′)), 4.11 (br, 1H,H—C(7′)), 2.91 (dd, J=16.2, 6.6 Hz, 1H, H—C(3′)), 2.31 (ddd, J=13.8,10.0, 3.7 Hz, 1H, H—C(2′)), 1.98-1.78 (m, 3H, H—C(2′), H—C(3′)), 1.07(s, 9H, (CH₃)₃—C—Si).

¹³C NMR (101 MHz, MeOD) δ 159.30 (C(2)), 155.14 (C(6)), 152.38 (C(4)),137.28 (C(8)), 136.93, 136.88 (CH-arom), 135.13, 134.78 (C-arom),131.07, 131.06, 128.91, 128.89 (CH-arom), 117.98 (C(5)), 87.72 (C(1′)),86.25 (C(4′)), 79.21, (C(7′)) 73.87 (C(5′)), 52.13 (C(3′)), 41.44(C(6′)), 38.35 (C(2′)), 27.42 ((CH₃)₃—C—Si), 19.82 ((CH₃)₃—C—Si)).

ESI⁺-HRMS m/z calcd for C₂₈H₃₄O₄N₅Si ([M+H]⁺) 532.2386, found 532.2367.

Example 50 (3′R,5′R,7′R)—N2-Acetyl-9-{5′-O-acetyl-7′-[(tert-butyldiphenylsilyl)oxy]-2′,3′-dideoxy-3′,5′-ethano-α-D-ribofuranosyl}guanine (50)

To a solution of nucleoside 49 (500 mg, 0.940 mmol) and4-Dimethylaminopyridine (276 mg, 2.4 mmol) in dry DCM (15 mL) was addedacetic anhydride (1.0 mL, 10.3 mmol) at rt. After stirring for 2 days,reaction was quenched by addition of satd NaHCO₃ (30 mL). The mixturewas then extracted with DCM (3×30 mL). The combined organic phases weredried over MgSO₄, filtered and evaporated. The crude product waspurified by CC (3.5% MeOH in DCM) to yield 50 (441 mg, 76%) as whitefoam.

Data for 50: R_(f)=0.62 (10% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 12.11 (br, 1H, NH—C(4)), 9.94 (br, 1H,H—N(1)), 7.62 (d, J=6.7 Hz, 5H, H-arom, H—C(8)), 7.46-7.31 (m, 6H,H-arom), 6.03 (dd, J=6.7, 2.7 Hz, 1H, H—C(1′)), 5.31 (dt, J=10.3, 5.2Hz, 1H, H—(C5′)), 4.99-4.81 (m, 1H, H—C(4′)), 4.02 (d, J=3.8 Hz, 1H,H—C(7′)), 2.88 (dd, J=16.0, 6.6 Hz, 1H, H—C(3′)), 2.44-2.20 (m, 4H,MeCONH, H—C(2′)), 2.12-1.73 (m, 6H, MeCO₂, H—C(6′), H—C(2′)), 1.04 (s,9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 172.73 (MeCONH), 170.46 (MeCO₂), 155.87(C(6)), 148.09 (C(4)), 147.47 (C(2)), 137.13 (C(8)), 135.74 (CH-arom),133.62, 133.29 (C-arom), 130.13, 130.09, 127.96, 127.93 (CH-arom),121.54 (C(5)), 86.47 (C(1′)), 82.81 (C(4′)), 76.60 (C(7′)), 74.37(C(5′)), 51.23 (C(3′)), 37.04, 37.01, (C(2′), C(6′)) 26.92((CH₃)₃—C—Si), 24.46 (MeCONH), 21.00 (MeCO₂), 19.05 ((CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₃₂H₃₈O₆N₅Si ([M+H]⁺) 616.2586, found 616.2580.

Example 51 (3'S,5′R,7′R)—N2-Acetyl-9-{5′-O-acetyl-2′,3′-dideoxy-3′,5′-ethano-7′-hydroxy-α-D-ribofuranosyl}guanine (51)

To a solution of nucleoside 50 (440 mg, 0.714 mmol) in dry THF (5 mL)was added TBAF (1M in THF, 1.1 mL, 1.1 mmol) at rt. The solution wasstirred for 4 hours at rt and then was directly purified by CC (13% MeOHin DCM) to yield 51 (235 mg, 87%) as white foam. Crystals suitable forX-ray analysis were obtained by recrystallization in a mixture ofH₂O/MeOH.

Data for 51: R_(f)=0.25 (13% MeOH in DCM);

¹H NMR (300 MHz, MeOD) δ 8.03 (s, 1H, H—C(8)), 6.28 (dd, J=7.0, 3.8 Hz,1H, H—C(1′)), 5.21 (ddd, J=9.2, 6.8, 5.1 Hz, 1H, H—C(5′)), 4.98 (dd,J=6.7, 5.0 Hz, 1H, H-(4′)), 4.13-4.05 (m, 1H, H—C(7′)), 3.17-3.05 (m,1H, H—C(3′)), 2.86 (ddd, J=13.8, 10.0, 3.8 Hz, 1H, H—C(2′)), 2.39-2.27(m, 1H, H—C(2′)), 2.24 (s, 3H, MeCONH), 2.16-2.00 (m, 5H, MeCO₂,H—C(6′)).

¹³C NMR (101 MHz, MeOD) δ 174.95 (MeCONH), 172.32 (MeCO₂), 157.50(C(6)), 149.96 (C(4)), 149.38 (C(2)), 139.66 (C(8)), 121.76 (C(5)),88.23 (C(1′)), 84.23 (C(4′)), 75.83 (C(5′), C(7′)), 51.65 (C(3′)),38.04, 37.93 (C(2′), C(6′)), 23.83 (MeCONH), 20.71 (MeCO₂).

ESI⁺-HRMS m/z calcd for C₁₆H₂₀O₆N₅ ([M+H]⁺) 378.1408, found 378.1419.

Example 52 (3'S,5′R,7′R)—N2-Acetyl-9-{5′-O-acetyl-2′,3′-dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}Guanine (52)

To a solution of the nucleoside 51 (186 mg, 0.492 mmol) in dry pyridine(10 mL) was added DMTr-Cl (501 mg, 1.48 mmol) at rt. The solution wasstirred for 2 days and then was diluted with satd NaHCO₃ (40 mL) andextracted with DCM (3×30 mL). The combined organic phases were driedover MgSO₄, filtered and evaporated. The crude product was purified byCC (3% MeOH in DCM, +0.5% Et₃N) to yield 52 (333 mg, 99%) as a yellowfoam.

Data for 52: R_(f)=0.56 (10% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 12.05 (br, 1H, NH—C(4)), 9.90 (br, 1H,H—N(1)), 7.40 (s, 1H, H—C(8)), 7.38-7.31 (m, 2H, H-arom), 7.28-7.08 (m,7H, H-arom), 6.75 (dd, J=9.0, 2.7 Hz, 4H, H-arom), 5.95-5.85 (m, 1H,H—C(1′)), 5.30-5.10 (m, 1H, H—C(5′)), 4.70-4.58 (m, 1H, H—C(4′)), 3.81(br, 1H, H—C(7′)), 3.68, 3.68 (2s, 6H, MeO), 2.25-2.07 (m, 5H, MeCONH,H—C(3′), H—C(2′)), 1.96-1.79 (m, 5H, MeCO₂, H—C(2′), H—C(6′)), 1.74-1.59(m, 1H, H—C(6′)).

¹³C NMR (75 MHz, CDCl₃) δ 172.65 (MeCONH), 170.42 (MeCO₂), 158.73,158.70 (MeO—C-arom), 155.86 (C(6)), 147.96 (C(4)), 147.43 (C(2)), 145.31(C-arom), 137.17 (C(8)), 136.69, 136.44 (C-arom), 130.32, 130.21,128.29, 128.05, 127.09 (CH-arom), 121.53 (C(5)), 113.38, 113.35(CH-arom), 87.25 (C(Ph)₃), 86.73 (C(1′)), 82.77 (C(4′)), 77.19 (C(7′)),74.37 (C(5′)), 55.38 (MeO-DMTr), 49.28 (C(3′)), 37.25 (C(2′)), 36.06(C(6′)), 24.40 (MeCONH), 21.01 (MeCO₂).

ESI⁺-HRMS m/z calcd for C₃₇H₃₈₀₈N5 ([M+H]⁺) 680.2715, found 680.2718.

Example 53(3'S,5′R,7′R)—N2-(N,N-Dimethylformamidino)-9-{2′,3′-dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}guanine (53)

To a solution of the nucleoside 52 (333 mg, 0.490 mmol) in dry MeOH (10mL) was added K₂CO₃ (305 mg, 2.20 mmol) at rt. The suspension wasstirred for 7 h at rt, then NH₄Cl (78 mg, 1.46 mmol) was added and theresulting mixture was filtered through a short pad of SiO₂. The SiO₂ waswashed with additional MeOH and then solvent was evaporated.

The crude product was dissolved in dry DMF (10 mL) andN,N-Dimethylformamide dimethyl acetal (0.33 mL, 2.5 mmol) was added. Thesolution was stirred for 2 hours at 55° C. and then the solvents wereremoved under reduced pressure. The crude product was purified by CC (7%MeOH in DCM, +0.5% Et₃N) to yield 53 (245 mg, 77%) as white foamcontaining traces of Et₃N.

Data for 53: R_(f)=0.32 (12% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.75 (br, 1H, H—N(1)), 8.25 (s, 1H,NCHN(CH₃)₂), 7.37 (d, J=7.3 Hz, 2H, H-arom), 7.29-7.08 (m, 8H, H-arom,H—C(8)), 6.74 (d, J=8.1 Hz, 4H, H-arom), 6.03 (dd, J=6.7, 2.8 Hz, 1H,H—C(1′)), 4.57 (dd, J=7.5, 4.6 Hz, 1H, H—C(4′)), 4.37-4.26 (m, 1H,H—C(5′)), 3.89 (t, J=3.9 Hz, 1H, H—C(7′)), 3.67, 3.67 (2s, 6H, MeO),3.24 (br, 1H, OH), 2.94 (s, 3H, NCHN(CH₃)₂), 2.87 (s, 3H, NCHN(CH₃)₂),2.35 (dd, J=15.9, 7.6 Hz, 1H, H—C(3′)), 1.94-1.68 (m, 4H, H—C(2′),H—C(6′)).

¹³C NMR (75 MHz, CDCl₃) δ 158.61 (MeO—C-arom), 158.28 (C(2)), 157.92(NCHN(CH₃)₂), 156.69 (C(6)), 149.90 (C(4)), 145.52, 136.86, 136.77(C-arom), 135.50 (C(8)), 130.15, 128.32, 127.92, 126.95 (CH-arom),120.27 (C(5)), 113.24 (CH-arom), 86.92 (C(Ph)₃), 85.57 (C(1′)), 85.12(C(4′)), 78.31 (C(7′)), 72.69 (C(5′)), 55.28 (MeO-DMTr), 49.28 (C(3′)),41.38 (NCHN(CH₃)₂), 39.77 (C(6′)), 37.58 (C(2′)), 35.04 (NCHN(CH₃)₂).

ESI⁺-HRMS m/z calcd for C₃₆H₃₉O₆N₆ ([M+H]⁺) 651.2926, found 651.2921.

Example 54(3'S,5′R,7′R)—N2-(N,N-Dimethylformamidino)-9-{5′-O-[(2-cyanoethoxy)-diisopropylaminophosphanyl]-2′,3′-dideoxy-3′,5′-ethano-7′-O-[(4,4′-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}guanine (54)

To a solution of the nucleoside 53 (245 mg, 0.377 mmol) and5-(Ethylthio)-1H-tetrazole (74 mg, 0.57 mmol) in dry DCM (15 mL) wasadded dropwise 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(0.20 mL, 0.64 mmol) at rt. After stirring for 50 min, the reactionmixture was diluted with satd NaHCO₃ (25 mL) and extracted with DCM(3×25 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (3% MeOH in DCM,+0.5% Et₃N) to yield 54 (212 mg, mixture of two isomers, 67%) as a whitefoam.

Data for 54: R_(f)=0.42 (7% MeOH in DCM);

¹H NMR (300 MHz, CDCl₃) δ 9.35 (br, 1H, H—N(1)), 8.51, 8.49 (2s, 1H,NCHN(CH₃)₂), 7.41-7.10 (m, 10H, H-arom, H—C(8)), 6.83-6.70 (m, 4H,H-arom), 6.15-6.00 (m, 1H, H—C(1′)), 4.64-4.36 (m, 2H, H—C(4′),H—C(5′)), 3.90-3.82 (m, 1H, H—C(7′)), 3.80-3.62 (m, 8H, MeO, OCH₂CH₂CN),3.59-3.43 (m, 2H, (Me₂CH)₂N), 3.04, 3.02 (2s, 6H, NCHN(CH₃)₂), 2.67-2.48(m, 2H, OCH₂CH₂CN), 2.32 (ddd, J=24.1, 15.1, 6.7 Hz, 1H, H—C(3′)),2.02-1.63 (m, 4H, H—C(2′), H—C(6′)), 1.14-1.03 (m, 12H, (Me₂CH)₂N).

¹³C NMR (101 MHz, CDCl₃) δ 158.76 (MeO—C-arom), 158.17, 158.12 (C(2)),158.03 (NCHN(CH₃)₂), 156.66, 156.59 (C(6)), 149.85, 149.79 (C(4)),145.51, 145.49, 136.84, 136.77, 136.73, 136.71 (C-arom), 135.76, 135.59(C(8)), 130.24, 130.20, 128.41, 128.33, 128.02, 127.10, 127.08(CH-arom), 120.74, 120.70 (C(5)), 117.98, 117.72 (OCH₂CH₂CN), 113.34(CH-arom), 87.16, 87.10 (C(Ph)₃), 86.00, 85.72 (C(1′)), 84.13, 84.10(J_(C,P)=3.6, 2.5 Hz, C(4′)), 78.02, 77.67 (C(7′)), 74.15, 73.74 (=15.3,18.7 Hz, C(5′)), 58.90, 58.67 (J_(C,P)=18.7, 19.7 Hz OCH₂CH₂CN), 55.38,55.36 (MeO-DMTr), 49.20, 49.09 (C(3′)), 43.20, 43.15 (J_(C,P)=12.4, 12.6Hz, ((Me₂CH)₂N), 41.42, 41.38 (NCHN(CH₃)₂), 38.68, 38.65 (C(6′)), 37.97,37.84 (C(2′)), 35.25 (NCHN(CH₃)₂), 24.83, 24.75, 24.68, 24.60, 24.53((Me₂CH)₂N), 20.35, 20.28 (OCH₂CH₂CN).

³¹P NMR (121 MHz, CDCl₃) δ 148.21, 148.01.

ESI⁺-HRMS m/z calcd for C₄₅H₅₆O₇N₈P ([M+H]⁺) 851.4004, found 851.4013.

Example 55(3aR,4R,6R,6aS)-4-((Tert-butyldiphenylsilyl)oxy)-2-methoxyhexahydro-2H-cyclopenta[b]furan-6-yl(4-nitrobenzoate) (55)

To a solution of the sugar 6 (195 mg, 0.437 mmol) and4-Dimethylaminopyridine (70 mg, 0.568 mmol) in dry DCM (10 mL) was added4-Nitrobenzoyl chloride (158 mg, 0.850 mmol) at rt. After stirringovernight, reaction was quenched by slow addition of satd NaHCO₃ (3 mL).The mixture was then diluted with satd NaHCO₃ (15 mL) and extracted withDCM (3×15 mL). The combined organic phases were dried over MgSO₄,filtered and evaporated. The crude product was purified by CC(EtOAc/hexanne 1:5) to yield a mixture of 55 (260 mg, 98%) in ananomeric ratio α/β≈4:1 as a white solid.

Data for 55: R_(f)=0.62 (EtOAc/hexane 1:2);

¹H NMR (300 MHz, CDCl₃) δ 8.33-8.17 (m, 4H, H-arom), 7.72-7.61 (m, 4H,H-arom), 7.51-7.32 (m, 6H, H-arom), 5.65-5.47 (m, 1H, H—C(6)), 4.97 (dd,J=9.2, 5.6 Hz, 1H, H—C(2)), 4.87 (t, J=5.8 Hz, 1H, H—C(6a)), 4.18 (d,J=5.0 Hz, 0.2H, H—C(4)), 3.98 (d, J=3.5 Hz, 0.8H, H—C(4)), 3.21 (d,J=15.1 Hz, 3H, MeO), 2.88 (dd, J=16.6, 7.9 Hz, 0.8H, H—C(3a)), 2.75-2.62(m, 0.2H, H—C(3a)), 2.49-2.34 (m, 0.2H, H—C(5)), 2.24-1.83 (m, 2.8H,H-(5), H—C(3)), 1.28 (ddd, J=13.0, 7.9, 4.9 Hz, 1H, H—C(3)), 1.09 (s,9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 164.46, 164.41 (CO₂R), 150.63 (O₂N—C-arom),135.87, 135.82 (CH-arom), 134.07, 133.75, 133.69 (CH-arom), 130.98,130.89, 129.98, 129.96, 129.91, 127.89, 127.87, 127.85, 123.59(CH-arom), 106.49, 106.39 (C(2)), 83.21, 79.87 (C(6a)), 76.54 (C(4)),76.09 (C(6)), 54.55, 54.47 (MeO), 51.69, 50.30 (C(3a), 38.07 (C(3)),37.17, 36.65 (C(5)), 27.04, 26.99 90 ((CH₃)₃—C—Si), 19.14 ((CH₃)₃—C—Si).

ESI⁺-HRMS m/z calcd for C₃₁H₃₅O₇NaSi ([M+Na]⁺) 584.2075, found 584.2085.

Example 56(3′R,5′R,7′R)-1-{7′-[(tert-butyldiphenylsilyl)oxy]-2,3′-dideoxy-3,5′-ethano-5′-O-(4-nitrobenzoate)-α,β-D-ribofuranosyl}Thymine (56)

To a solution of the sugar 55 (260 mg, 0.463 mmol) and thymine (84 mg,0.695 mmol) in dry MeCN (3 mL) was added dropwise BSA (0.34 mL, 1.4mmol) at rt. After stirring for 30 min at rt, the solution was cooleddown to 0° C. and TMSOTf (0.10 mL, 1.3 mmol) was added dropwise. Afterfurther stirring for 2 h at 0° C. and for 18 h at rt, the reactionmixture was diluted with satd NaHCO₃ (30 mL) and extracted with DCM(4×40 mL). The combined organic phases were dried over MgSO₄, filteredand evaporated. The crude product was purified by CC (2% MeOH in DCM) toyield a mixture of 56 (240 mg, 79%) in an anomeric ratio α/β≈88:12 aswhite foam.

Data for 56: R_(f)=0.56 (DCM+3% MeOH);

¹H NMR (300 MHz, CDCl₃) δ 9.38 (br, 1H, (s, 1H, H—N(3)), 8.32-8.23 (m,2H, H-arom), 8.22-8.11 (m, 2H, H-arom), 7.65 (dd, J=7.7, 1.5 Hz, 4H,H-arom), 7.50-7.36 (m, 6H, H-arom), 6.95 (d, J=0.9 Hz, 1H, H—C(6)), 5.96(t, J=6.3 Hz, 1H, H—C(1′)), 5.55 (dt, J=9.9, 6.0 Hz, 1H, H—C(5′)), 5.13(dd, J=6.4, 5.4 Hz, 1H, H—C(4′)), 4.20-4.05 (m, 1H, H—C(7′)), 2.94-2.78(m, 1H, H—C(3′)), 2.22 (dd, J=13.3, 6.4 Hz, 1H, H—C(6′)), 2.09-1.73 (m,6H, H—C(6′), H—C(2′), Me-C(5)), 1.09 (s, 9H, (CH₃)₃—C—Si).

¹³C NMR (75 MHz, CDCl₃) δ 164.32, 163.79 (C(4), CO₂R), 150.65, 150.39(O₂N—C-arom, C(2)), 135.70, 135.68 (CH-arom), 135.13 (C-arom), 134.83(C(6)), 133.46, 133.10 (C-arom), 130.91, 130.73, 130.11, 127.93, 123.60(CH-arom), 111.30 (C(5)), 87.26 (C(1′)), 82.44 (C(4′)), 76.40 (C(7′)),76.07 (C(5′)), 50.76 (C(3′)), 37.94 (C(6′)), 36.68 (C(2′)), 26.89((CH₃)₃—C—Si), 19.03 ((CH₃)₃—C—Si), 12.62 (Me-C(5)).

ESI⁺-HRMS m/z calcd for C₃₅H₃₇O₈N₃NaSi ([M+Na]⁺) 678.2242, found678.2254.

Example 57(3′R,5′R,7′R)-1-{2′,3′-dideoxy-3′,5′-ethano-7′-hydroxy-5′-O-(4-nitrobenzoate)-α,β-D-ribofuranosyl}thymine (57)

To a solution of the nucleoside 56 (220 mg, 0.335 mmol) in dry THF (2mL) was added TBAF (1M in THF, 0.84 mL, 0.84 mmol) at rt. After stirringfor 4 h at rt, the reaction mixture was diluted with satd NaHCO₃ (20 mL)and extracted with EtOAc (3×20 mL) and DCM (2×80 mL). The combinedorganic phases were dried over MgSO₄, filtered and evaporated. The crudeproduct was purified by CC (5% MeOH in DCM) to yield an anomeric mixtureof 57 (101 mg, 72%). Crystals suitable for X-ray analysis were obtainedby recrystallization in EtOAc.

Data for 57: R_(f)=0.50 (DCM+7% MeOH);

¹H NMR (300 MHz, CDCl₃) δ 8.96 (br, 1H, H—N(3)), 8.34-8.17 (m, 4H,H-arom), 7.07 (d, J=1.1 Hz, 1H, H—C(6)), 6.11 (t, J=6.3 Hz, 1H,H—C(1′)), 5.57-5.45 (m, 1H, H—C(5′)), 5.15 (dd, J=6.6, 5.4 Hz, 1H,H—C(4′)), 4.38-4.23 (m, 1H, H—C(7′)), 2.96 (dd, J=13.5, 6.9 Hz, 1H,H—C(3′)), 2.26 (ddd, J=13.1, 10.3, 5.4 Hz, 4H, H—C(2′), H—C(6′)), 1.91(d, J=0.9 Hz, 3H, Me-C(5)).

ESI⁺-HRMS m/z calcd for C₁₉H₁₉O₈N₃Na ([M+Na]⁺) 440.1064, found 440.1072.

Example 58 Design and Synthesis of Oligomers of Alpha Anomeric Monomers

To assess the accommodation of the modification inside natural β-DNAstrand, five oligodeoxynucleotides (ON16-20) with single or multipleinsertions of the thymidine building block 6 were prepared. In order tofit the geometry of β-DNA, the modification was inserted with polarityreversal, resulting in 3′-7′ and 5′-′5 nucleosidic linkages (see FIGS. 1and 4). To test the pairing properties of this new system with naturalnucleic acid, but also toward itself, the fully modified ON21 containingall four nucleobases, as well as its antiparallel (ON22) and parallel(ON23) fully modified complements were prepared. Finally, a fullymodified strand with phosphorothioate linkage was synthesized (ON24),with the aim to test its potential antisense properties. The syntheseswere performed using classical automated phosphoramidite chemistry.Fully modified strands were synthesized in a 5′→7′ direction. As aconsequence, complete cleavage of the DMTr protecting group from the 7′position required a solution of 5% dichloroacetic acid indichloroethane. In these conditions, coupling yield were >98% based ontrityl assay. Fully modified strands could be completely cleaved fromuniversal solid support by a smooth treatment in concentrated ammonia at55° C. overnight (for further synthetic and analytical details seebelow).

Process of Alpha Anomeric Oligonucleotide Synthesis, Deprotection andPurification

Oligonucleotides syntheses were performed on aPharmaci-Gene-Assembler-Plus DNA synthesizer on a 1.3 μmol scale.Natural DNA phosphoramidites (dT, dC4bz, dG2DMF, dA6Bz) and solidsupport (dA-Q-CPG 500, dmf-dG-Q-CPG 500, Glen Unysupport 500) werepurchased from Glen Research. Natural DNA phosphoramidites were preparedas a 0.1 M solution in MeCN and were coupled using a 4 min step.7′,5′-α-bc-DNA phosphoramidites were prepared as a 0.1 M solution in1,2-dichloroethane and were coupled using an extended 12 min step.5-(Ethylthio)-1H-tetrazole (0.25 M in MeCN) was used as coupling agent.Detritylation of modified nucleoside was performed with a solution of 5%dichloroacetic acid in dichloroethane. Sulfurization was performed witha solution of 0.2 M phenylacetyl disulfide in MeCN/pyridine (1:1) andwith a reaction time of 3.5 min Capping and oxidation were performedwith standard conditions. Cleavage from solid support and deprotectionof oligonucleotides was achieved by treatment with concentrated ammoniaat 55° C. for 16 h. After centrifugation, the supernatant werecollected, the beads further washed with H₂O (0.5 mL×2) and theresulting solutions were evaporated to dryness. Crude oligonucleotideswere purified by ion-exchange HPLC (Dionex-DNAPac PA200). Buffersolutions of 25 mM Trizma in H₂O, pH 8.0, was used as the mobile phase“A” and 25 mM Trizma, 1.25 M NaCl in H₂O, pH 8.0, was used as the mobilephase “B”. For the phosphorothioate strand, a buffer solution of 10 mMNaOH in H₂O, pH 12.0, was used as the mobile phase “A” and 10 mM NaOH,2.50 M NaCl in H₂O, pH 12.0, was used as the mobile phase “B”. Thepurified oligonucleotides were then desalted with Sep-pack C-18cartridges. Concentrations were determined by measuring the absorbanceat 260 nm with a Nanodrop spectrophotometer, using the extinctioncoefficient of the corresponding natural DNA oligonucleotides.Characterizations of oligonucleotides were performed by ESI⁻ massspectrometry or by LC-MS.

Example 59 Pairing Properties of Modified OligodeoxynucleotidesSynthesized of Alpha Anomeric Monomers with Complementary DNA and RNA

The duplex's stability of oligonucleotides was assessed by UV meltingcurves at 260 nm, and their T_(m)s were compared to their natural DNAanalogs (Table 1). Oligonucleotides ON16-17 with a single incorporationresulted in a strong destabilization with DNA complements and slightlylower destabilization with RNA. This penalty appears to be cumulativeand ON18, with the two previous positions modified, further decreasedthe T_(m)s. However, when two or five modifications were introducedconsecutively (ON19-20), the destabilization per modification is reducedto approximately −3° C. versus DNA and −1.3° C. versus RNA. These datasuggests that a junction between the DNA and 7′,5′-α-bc-DNA induces astrong destabilization, with a depreciation of T_(m) between −4 and −9°C., depending on the sequence context. Such destabilizations byheterobackbone junctions have already been observed for α-DNA (Araminiet al., Nucleic Acids Res 1998, 26, 5644, Aramini et al., Biochemistry1997, 36, 9715) and for α-LNA (Nielsen et al., Chemistry—A EuropeanJournal 2002, 8, 712). This destabilization could be compensated byinserting the modifications as a block.

TABLE 1T_(m) and ΔT_(m)/mod data from UV-melting curves (260 nm) of ON16-20 induplex with complementary DNA and RNA. T_(m)(° C.) ΔT_(m)/modT_(m)(° C.) ΔT_(m)/mod Entry Sequence^(a,b,c) vs DNA (° C.) vs RNA(° C.) ON16 5′-d(GGA TGT TCt CGA)-3′ 40.0 -9.1 42.6 -6.8 (SEQ ID NO: 16)ON17 5′-d(GGA t GT TCT CGA)-3′ 43.0 -6.1 45.8 -3.6 (SEQ ID NO: 17) ON185′-d(GGA tGT TCt CGA)-3′ 32.8 -8.1 38.0 -5.7 (SEQ ID NO: 18) ON195′-d(GGA TGt tCT CGA)-3′ 42.9 -3.1 47.0 -1.2 (SEQ ID NO: 19) ON205′-d(GCA ttt ttA CCG)-3′^(d) 34.0 -2.7 37.2 -1.4 (SEQ ID NO: 20)^(a)total strand conc. 2 μM in 10 mM NaH₂PO₄, 150 mM NaCl, pH 7.0.^(b)A, G, T, C denote natural 2′-deoxynucleosides; t (bold type)corresponds to modified nucleosides. ^(c)T_(m) of unmodified duplexes,DNA/DNA: 49.1° C., DNA/RNA: 49.4° C. ^(d)T_(m) of unmodified duplexes,DNA/DNA: 47.5° C., DNA/RNA: 44.0° C.

Example 60 Pairing Properties of Fully Modified OligonucleotidesSynthesized of Alpha Anomeric Monomers

As expected, all three fully modified sequences ON21-23 exhibit acooperative and reversible melting behavior with their parallel DNA andRNA complements (FIG. 5), but not with their antiparallel complements.The resulting 7′,5′-α-bc-DNA/DNA duplexes are slightly less stable thantheir natural counterparts, with a destabilization between −0.1 and−0.5° C. per modification (Table 2). Surprisingly, it has been foundthat ON21-23 form very stable duplexes with RNA, resulting in a greatstabilization between 1.3 and 1.5° C. per modification. A quiteastonishing difference exists on the stabilizing effect between thefully modified strands and natural oligodeoxynucleotides strands withsingle or multiple incorporations. This stresses the importance ofsynthesizing fully modified strands in order to characterize a newpairing system.

TABLE 2T_(m) and ΔT_(m)/mod data from UV-melting curves (260 nm) of ON21-24 induplex with complementary parallel DNA and RNA. T_(m)(° C.) T_(m)(° C.)vs  vs parallel ΔT_(m)/mod parallel ΔT_(m)/mod Entry Sequence^(a,b) DNA(° C.) RNA (° C.) ON21 5′-d(agc tct tgt agg)-7′^(c) 43.2 -0.5 65.0 1.3(SEQ ID NO: 21) ON22 5′-d(cct aca aga gct)-7′^(d) 43.8 -0.4 58.6 1.3(SEQ ID NO: 22) ON23 5′-d(tcg aga aca tcc)-7′^(e) 47.7 -0.1 61.6 1.5(SEQ ID NO: 23) ON24 5′-d(t*c*c*a*t*t*c*g*g*c*t*c*c*a*a*)-7′^(f) 43.2-1.3 77.0 0.6 (SEQ ID NO: 24) ^(a)total strand conc. 2 μM in 10 mMNaH₂PO₄, 150 mM NaCl, pH 7.0. ^(b) a, g, t, c corresponds to modifiedadenine, guanine, thymine and methylcytosine respectively, * denotes aphosphorothioate linkage. ^(c)T_(m) of unmodified duplexes, DNA/DNA:49.1° C., DNA/RNA: 49.4° C. ^(d)T_(m) of unmodified duplexes, DNA/RNA:43.0° C. ^(e)T_(m) of unmodified duplexes, DNA/DNA: 49.0° C., DNA/RNA:43.3° C. ^(f)T_(m) of unmodified duplexes, DNA/DNA: 62.0° C., DNA/RNA:67.4° C.

To test the mismatches discrimination, UV melting experiments wereperformed with ON21 and its parallel DNA complements possessing allthree alternative nucleobases at the position 4 (Table 3). Suchmispairing has a strong destabilizing effect and reduce the T_(m)s by−9.6 to −14.3° C. When compared to its natural counterpart,7′,5′-α-bc-DNA has a better mismatch discrimination ability with theT_(m)s further reduced by −1.0 to −2.4° C. Increasing the mismatchdiscrimination should reduce the potential off-target effects andtherefore represents an appealing property for an antisense candidate.Modification with a phosphorothioate linkage is well accommodated in thecontext of 7′,5′-α-bc-DNA and the destabilizing effect is in the rangeof the −0.5° C. per nucleotide reported for this modification (Kurreck,J. European journal of biochemistry/FEBS 2003, 270, 1628). ON24maintains a good affinity toward RNA with a stabilizing effect of 0.6°C. when compared with natural DNA.

TABLE 3 T_(m) values from UV-melting curves (260 nm) of ON21 and DNA1 induplex with complementary DNA incorporating one mismatch. EntryDuplex^(a) X = A X = T X = G X = C ON21 5′-d(agc tct tgt agg)-7′ 43.230.0 33.5 28.9 (SEQ ID NO: 21) DNA-X 5′-d(TCG XGA ACA TCC)-3′(SEQ ID NO: 25) DNA1 5′-d(GGA TGT TCT CGA)-3′ 49.1 38.3 40.5 36.7(SEQ ID NO: 26) DNA-X 5′-d(TCG XGA ACA TCC)-3′ (SEQ ID NO: 25)Experimental conditions: total strand conc. 2 μM in 10 mM NaH₂PO₄, 150mM NaCl, pH 7.0 ^(a)A, G, T, C denote natural 2′-deoxynucleosides; a, g,t, c corresponds to modified adenine, guanine, thymine andmethylcytosine respectively.

In its own series, ON21 formed a very stable duplex toward itsantiparallel complement ON22, resulting in an unexpected T_(m) of 83.6°C. Due to this high T_(m), the complete classical sigmoidal transitioncould be observed only in the absence of sodium chloride, decreasing theT_(m) to 68.6° C. (Table 4). Interestingly, the formation of duplexresulted in a low hypochromicity of only 10% (FIG. 5). This is anindication of a base stacking differing from classical helix andtherefore, formation of duplex with a geometry deviating from canonicalA or B-duplexes could be expected. On the other hand, no sigmoidalmelting transition has been observed between ON21 and its parallelcomplement ON23 (FIG. 5). The change in hypochromicity occurring doesnot differ from the UV melting experiments performed on the two singlestrand separately, which was associated with a base stacking occurringinside the single strands. To test the ability of 7′,5′-α-bc-DNA to fitinside A-like helix, a melting experiment was performed towardtricyclo-DNA (tc-DNA), a conformationally constrained mimic of RNA(Renneberg et al., Journal of the American Chemical Society 2002, 124,5993.). When ON21 is mixed with complementary parallel tc-DNA strand(Tc1), a surprisingly high Tm of 81° C. was observed, demonstrating theability of 7′,5′-α-bc-DNA to adapt to this helix geometry.

TABLE 4 T_(m) values from UV-melting curves (260 nm) of ON22-23 andTc1 in duplex with ON21. NaCl con- centration ΔT_(m) (° C.) EntrySequence^(a,b) [m/M] VS ON21 ON22 5′-d(cct aca aga gct)-7′ 150  83.6(SEQ ID NO: 22) ON22 5′-d(cct aca aga gct)-7′  50  79.6 (SEQ ID NO: 22)ON22 5′-d(cct aca aga gct)-7′   0  68.6 (SEQ ID NO: 22) ON23 5′-d(tcg aga aca tcc )-7′ 150 <10 (SEQ ID NO: 23) Tel 5′-d( tcg aga   aca  tcc )-3′ 150  81.0 (SEQ ID NO: 27) ^(a)total strand conc. 2 μM in 10 mMNaH₂PO₄, pH 7.0. ^(b)a, g, t, c corresponds to modified adenine,guanine, thymine and methylcytosine respectively, a , g , t , ccorresponds to tricyclo-DNA adenine, guanine, thymine and methylcytosinerespectively

Example 61 Thermodynamic Data of Duplex Formation of Alpha AnomericOligomers

The thermodynamic data for duplexes formations of ON21 with DNA and RNAand their natural counterpart have been extracted by curves fitting tothe experimental melting curves, following an established methodology(Petersheim et al., Biochemistry 1983, 22, 256) (Table 5). As expected,the free energy ΔG at 25° C. follows the same trend as the T_(m) data,with ON21.RNA being the most favored duplex. With respect to naturalsystem, ON21 binds with natural nucleic acids with a lower enthalpy.However, this destabilization is compensated by an entropic gain. Thisbehavior is typical in the bc-DNA series and arises from theconformational rigidity added by the ethylene bridge. Interestingly, theselectivity of 7′,5′-α-bc-DNA for RNA over DNA is mostly enthalpicallydriven.

TABLE 5 Thermodynamic data of duplex formation. ΔH ΔS ΔG25° C. [kcal[cal mo1⁻¹ [kcal Duplex Sequences^(a) mol⁻¹]  K⁻¹] mol⁻¹] DNA•DNA5′-GGA TGT TCT CGA-3′ -91.7 -257.9 -14.8 (SEQ ID NO: 26)3′-CCT ACA AGA GCT-5′ (SEQ ID NO: 29)\ DNA•RNA 5′-GGA TGT TCT CGA-3′-92.1 -258.3 -15.0 (SEQ ID NO: 26) 3′-CCU ACA AGA GCU-5′ (SEQ ID NO: 28)ON21•DNA 7′-gga tgt tct cga-5′ -79.7 -224.2 -12.9 (SEQ ID NO: 21)3′-CCT ACA AGA GCT-5′ (SEQ ID NO: 29) ON21•RNA 7′-gga tgt tct cga-5′-83.9 -222.0 -17.7 (SEQ ID NO: 21) 3′-CCU ACA AGA GCU-5′(SEQ ID NO: 28), ^(a)A, G, T, U, C denote nucleosides; a, g, t, ccorresponds to modified adenine, guanine, thymine and methylcytosinerespectively

Example 62 CD-Spectroscopy of Alpha Anomeric Oligomer

The CD-spectra of ON21 in duplex with DNA, RNA or ON22 have beenmeasured and compared with the corresponding natural DNA/RNA duplex(FIG. 6). Both duplexes of ON21 with DNA or RNA have a CD signaturerelatively close the natural A/B-helix. However, the ON21/DNA duplexdoes not display a negative signal at 210 nm and has the ellipticity at226 nm blue shifted by 5 nm and associated with a gain in amplitude. TheON21/RNA duplex also has a peak of higher positive amplitude at 226 nm,and the positive ellipticity at 266 is blue shifted by 4 nm and formed asharper peak. On the other hand, the modified homo duplex has a veryatypical CD signature, characterized by a broad negative ellipticitybetween 275 and 300 nm of small amplitude and two positive peaks at 259nm and 218 nm. In agreement with low hypochromicity change upon duplexformation, the CD spectra of the homo duplex indicate the formation of astructure deviating from conventional helix.

Example 63 Biological Stability of Alpha Anomeric Oligomers

The biostability of the fully modified oligonucleotide ON21 was studiedunder simulated physiological conditions in comparison with itscorresponding natural oligonucleotide. Oligonucleotides were incubatedin a 1:1 mixture of H₂O and human serum at 37° C. and reaction outcomewere analyzed by 20% denaturing PAGE. In detail, ON21 and itscorresponding natural oligonucleotide were diluted to 10 μM in a 1:1mixture of H₂O and human serum (from human male AB plasma, USA origin,sterile-filtered (Sigma)). The reactions were performed at a 20 μL scaleand were incubated at 37° C. Control reactions (a, were performed byincubating the oligonucleotides at 10 μM in H₂O at 37° C. for 24 hours.The reactions were stopped at specific times by addition of formamide(20 μL). The resulting mixtures were stored at −20° C. before being heatdenaturated for 5 min at 90° C. and then analyzed by 20% denaturingPAGE. Visualization was performed with a stains-all solution. Theexperiment showed complete digestion of natural DNA strand already after4 hours (d), where the modified oligonucleotide remained completelystable even after 24 hours (j) (FIG. 7). The 7′,5′-α-bc-DNA modificationappears to confer a significantly improved biostability.

Example 64 Complement C3 Activation by Alpha Anomeric Oligomers

Complement activation represents an important toxic response oftenassociated with the in vivo use of antisense ONs. Moreover, in vivotests performed with tc-DNA induced occasionally such an acute toxicity,limiting consequently their use. In this context, it is of particularinterest to test the complement activation of 7′,5′-α-bc-DNA, containingphosphorothioate nucleosidic linkages, and compare it withwell-characterized modified or natural ONs. Experiments were carried byincubating mouse serum samples with 4 mg/ml of the tested ONs at 37° C.for 45 min. Mouse C3 complement activation was then analyzed by ELISAusing PanSpecific C3 reagent followed by SC5b-9 kit.

Incubation with ON24 (PS-7′,5′-α-bc-DNA scaffold) resulted in level ofC3 complement protein lower than with natural DNA, but higher than withPS-DNA (FIG. 8). The protein level is similar than with non-toxicPO-tc-DNA. These promising results indicate that ON24 does not activatethe complement significantly.

Example 65 Antisense Activity

Duchenne muscular dystrophy (DMD) is a lethal muscle degenerativedisease that arises from mutations in the DMD gene resulting inout-of-frame dystrophin transcripts and ultimately in the lack offunctional dystrophin protein. In DMD, aberrant disease-related pre-mRNAtranscripts can be functionally restored by antisense oligonucleotides(AO). Such AOs can change the slice pattern and can correct aberrant outof frame dystrophin transcripts via the exclusion of specific dystrophinexons. Thereby the open reading frame is restored and a shortened butfunctional dystrophin protein product is generated (Yang et al., PloSone 2013, 8, e61584). The ability of the 7′,5′-α-bc-DNA scaffold toinduce exon skipping was assessed in vitro by transfecting myoblastsfrom mdx mice—a murine model for Duchenne muscular dystrophy—with ON24using lipofectamine LTX. Mdx myoblasts were incubated with7′,5′-α-bc-DNA, and RNA was isolated, amplified by nested RT-PCR andanalyzed by gels (PAGE).

The results indicate a high level of exon 23 skipping, as well as asignificant level of a double skip of exons 22 and 23 (FIG. 9). Thisdouble exons skipping is often encountered for compounds efficient inrestoring disrupted reading frames (Mann et al., Proceedings of theNational Academy of Sciences of the United States of America 2001, 98,42; Mann et al., The journal of gene medicine 2002, 4, 644; Yin et al.,J. Molecular therapy. Nucleic acids 2013, 2, e124; Yang et al., PloS one2013, 8, e61584) and testifies to the potency of ON24 for therapeuticexon skipping in muscular dystrophies. Moreover, 7′,5′-α-bc-DNA induceda higher level of exon skipping than tc-DNA, a modification known tohave a significant therapeutic effect in mice (Goyenvalle et al., NatMed 2015, 21, 270). Therefore, the 7′,5′-α-bc-DNA scaffold meets theprerequisites to induce a strong effect in therapeutic exon skipping.

Example 66 X-Ray Crystal Structure Determination of Alpha-Anomers

Crystals of the monomers were obtained, mainly to confirm the relativeconfiguration of the 7′,5′-α-bc-DNA series, but also to compare thisstructure with the minimum found by ab initio calculations. To obtaincrystals for the thymidine and the guanosine monomers, a p-nitrobenzoatewas introduced at O3′ to be able to crystalize the T monomers (compound57). This molecule co-crystalized with EtOAc giving rise to crystals oflow quality. The resulting structure (FIG. 3a ) adopts a C1′-endo,O4′-exo sugar pucker and a C6′-endo conformation in the carbocyclicring. This conformation orients the C5′ substituent in apseudoequatorial position and the C7′ hydroxyl group in a pseudoaxialposition. This structure deviates in the sugar pucker from the minimapredicted by ab initio calculations. However, further analysisdemonstrated that the p-nitrobenzoate substituent disturbed the sugarconformation. The protected guanosine 51 gave rise to crystals of goodquality. In this case (FIG. 3b ), the furanose adopts an almost perfectC1′-exo conformation, while the carbocyclic ring adopts a geometry asdescribed above. This time, the structure matches perfectly with one ofthe minimal conformers predicted by ab initio calculation.

Compound 57:

A colorless transparent crystal of [C₁₉H₁₉N₃O₈]₂[0.5(C₄H₈O₂)] wasmounted in air and used for X-ray structure determination at ambientconditions. All measurements were made on an Oxford DiffractionSuperNova area-detector diffractometer (Dupradeau et al., Nucleic AcidsRes 2008, 36, D360) using mirror optics monochromated Mo Kα radiation(λ=0.71073 Å) and Al filtered (Lu et al., Nature protocols 2008, 3,1213). The unit cell constants and an orientation matrix for datacollection were obtained from a least-squares refinement of the settingangles of reflections in the range 1.7°<θ<28.07°. A total of 440 frameswere collected using co scans, with 15+15 seconds exposure time, arotation angle of 1° per frame, a crystal-detector distance of 65.1 mm,at T=223(2) K. Data reduction was performed using the CrysAlisProprogram (Dupradeau et al., Nucleic Acids Res 2008, 36, D360). Theintensities were corrected for Lorentz and polarization effects, and anabsorption correction based on the multi-scan method using SCALE3ABSPACK in CrysAlisPro was applied. Data collection and refinementparameters are given in Table 6. The structure was solved by directmethods using SHELXT, which revealed the positions of all non-hydrogenatoms of the title compound. The non-hydrogen atoms were refinedanisotropically. All H-atoms were placed in geometrically calculatedpositions and refined using a riding model where each H-atom wasassigned a fixed isotropic displacement parameter with a value equal to1.2 Ueq of its parent atom. Refinement of the structure was carried outon F² using full-matrix least-squares procedures, which minimized thefunction Σw(F_(o) ²−F_(c) ²)². The weighting scheme was based oncounting statistics and included a factor to downweight the intensereflections. All calculations were performed using the SHELXL-2014/7program. The compound crystallizes in the monoclinic space group C 2,with a monoclinic angle very close to 90 degrees, which implies an easypseudo-merohedral twinning that cannot be easily deconvoluted. Moreover,the p-NO₂-benzoate group of the main molecule is disordered over twoconformation and the co-crystallized acetate solvent is disordered abouta twofold axis. For all these reasons, the structure determination andrefinement was somewhat complicated, some short intermolecular contactsare calculated, and the absolute configuration cannot be determined(Flack parameter being unrealistic), but it is assigned according to thereaction sequence.

TABLE 6 Crystal data and structure refinement for compound 57Identification code shelx Empirical formula C21 H23 N3 O9 Formula weight461.42 Temperature 223(2) K Wavelength 0.71073 Å Crystal systemMonoclinic Space group C 2 Unit cell dimensions a = 22.6224(5) Å a = 90°b = 7.9610(2) Å b = 90.172(2)° c = 12.0447(3) Å g = 90° Volume2169.20(9) Å3 Z 4 Density (calculated) 1.413 Mg/m³ Absorptioncoefficient 0.112 mm⁻¹ F(000) 968 Crystal size 0.344 × 0.265 × 0.072 mm³Theta range for data 1.691 to 28.071°. collection Index ranges −28 <= h<= 28, −9 <= k <= 10, −15 <= l <= 15 Reflections collected 7292Independent reflections 4360 [R(int) = 0.0206] Completeness to 100.0%theta = 25.242° Absorption correction Gaussian Max. and min. 0.994 and0.979 transmission Refinement method Full-matrix least- squares on F2Data/restraints/ 4360/319/405 parameters Goodness-of-fit on F2 1.027Final R indices R1 = 0.0541, wR2 = 0.1280 [I > 2sigma(I)] R indices (alldata) R1 = 0.0694, wR2 = 0.1414 Absolute structure 1.7(6) parameterExtinction coefficient n/a Largest diff. peak 0.329 and −0.208 e.Å−3 andhole

Compound 51:

A colorless transparent crystal of [C16H19N5O6].(CH4O) was mounted inair and used for X-ray structure determination at ambient conditions.All measurements were made on an Oxford Diffraction SuperNovaarea-detector diffractometer (Dupradeau et al., Nucleic Acids Res 2008,36, D360) using mirror optics monochromated Mo Kα radiation (λ=0.71073Å) and Al filtered. The unit cell constants and an orientation matrixfor data collection were obtained from a least-squares refinement of thesetting angles of reflections in the range 1.5°<θ<27.2°. A total of 970frames were collected using w scans, with 45+45 seconds exposure time, arotation angle of 1° per frame, a crystal-detector distance of 65.1 mm,at T=123(2) K. Data reduction was performed using the CrysAlisProprogram. The intensities were corrected for Lorentz and polarizationeffects, and an absorption correction based on the multi-scan methodusing SCALES ABSPACK in CrysAlisPro was applied. Data collection andrefinement parameters are given in Table 7. The structure was solved bydirect methods using SHELXS-97, which revealed the positions of allnon-hydrogen atoms of the title compound. The non-hydrogen atoms wererefined anisotropically. All H-atoms were placed in geometricallycalculated positions and refined using a riding model where each H-atomwas assigned a fixed isotropic displacement parameter with a value equalto 1.2 Ueq of its parent atom. Refinement of the structure was carriedout on F² using full-matrix least-squares procedures, which minimizedthe function Σw(F_(o) ²−F_(c) ²)². The weighting scheme was based oncounting statistics and included a factor to downweight the intensereflections. All calculations were performed using the SHELXL-97 program(Lu et al., Nature protocols 2008, 3, 1213).

TABLE 7 Crystal data and structure refinement for compound 51 Empiricalformula C17H23N5O7 Formula weight 409.40 Temperature 123(2) K Wavelength0.71073 Å Crystal system Monoclinic Space group P 21 Unit celldimensions a = 4.83370(10) Å a = 90° b = 25.1367(4) Å b = 92.4850(10)° c= 15.2979(2) Å g = 90° Volume 1857.00(5) Å3 Z 4 Density (calculated)1.464 Mg/m³ Absorption coefficient 0.115 mm⁻¹ F(000) 864 Crystal size0.4678 × 0.1255 × 0.0405 mm³ Theta range for data 1.559 to 27.205°collection Index ranges −6 <= h <= 6, −31 <= k <= 32, −19 <= l <= 19Reflections collected 25075 Independent reflections 7480 [R(int) =0.0452] Completeness to 100.0% theta = 25.000° Absorption correctionGaussian Max. and min. 0.996 and 0.97 transmission Refinement methodFull-matrix least- squares on F2 Data/restraints/ 7480/1/533 parametersGoodness-of-fit on F2 1.058 Final R indices R1 = 0.0579, [I > 2sigma(I)]wR2 = 0.1456 R indices (all data) R1 = 0.0657, wR2 = 0.1518 Absolutestructure 0.7(4) parameter Extinction coefficient n/a Largest diff. peak1.403 and −0.492 e.Å−3 and hole

Example 67 Design and Synthesis of Oligomers of Beta Anomeric Monomers

A series of oligonucleotides with single and multiple incorporations ofbuilding blocks 12, 14, 19 and 25 as well as fully modified sequenceswere synthesized using classical automated phosphoramidite chemistry(for synthetic and analytical details see suppl. Inf.). Oligonucleotideswith single and multiple incorporations were primarily prepared todetermine the effect of the modifications on the T_(m) when complexedwith complementary DNA and RNA, as well as to determine the Watson-Crickbase pairing selectivity. Fully modified oligonucleotides weresynthesized to characterize the pairing behavior not only with naturalDNA and RNA, but also to investigate the self-pairing of 7′,5′-bc-DNA.The main interest here was to determine whether this novel structuralDNA analogue is capable of forming an independent base-pairing systemthat could be of interest as an alternative genetic system.

Process of Beta Anomeric Oligonucleotide Synthesis, Deprotection andPurification

Oligonucleotides syntheses were performed on aPharmaci-Gene-Assembler-Plus DNA synthesizer on a 1.3 μmol scale.Natural DNA phosphoramidites (dT, dC^(4bz), dG^(2DMF), dA^(6Bz)) andsolid support (dA-Q-CPG 500, dmf-dG-Q-CPG 500, Glen Unysupport 500) werepurchased from Glen Research. Natural DNA phosphoramidites were preparedas a 0.1 M solution in MeCN and were coupled using a 4 min step. Bc-DNAphosphoramidites were prepared as a 0.1 M solution in 1,2-dichloroethaneand were coupled using an extended 12 min step.5-(Ethylthio)-1H-tetrazole (0.25 M in MeCN) was used as coupling agent.Capping, oxidation and detritylation were performed with standardconditions. Cleavage from solid support and deprotection ofoligonucleotides was achieved by treatment with concentrated ammonia at55° C. for 16 h. After centrifugation, the supernatant were collected,the beads further washed with H₂O (0.5 mL×2) and the resulting solutionswere evaporated to dryness. Fully modified sequences were furthertreated with NaOH (0.4 M in H₂O/MeOH 1:1) for 1 h at rt to achievecomplete cleavage from the universal support linker and were thenfiltrated using spin-columns (Amicon Ultra 0.5 mL centrifugal filters,MWCO 3 kDa). Crude oligonucleotides were purified by ion-exchange HPLC(Dionex-DNAPac PA200). Buffer solutions of 25 mM Trizma in H₂O, pH 8.0,was used as the mobile phase “A” and 25 mM Trizma, 1.25 M NaCl in H₂O,pH 8.0, was used as the mobile phase “B”. The purified oligonucleotideswere then desalted with Sep-pack C-18 cartridges. Concentrations weredetermined by measuring the absorbance at 260 nm with a Nanodropspectrophotometer, using the extinction coefficient of the correspondingnatural DNA oligonucleotides. Characterizations of oligonucleotides wereperformed by ESI⁻ mass spectrometry.

Example 68 Pairing Properties of Modified β-Oligodeoxynucleotides withComplementary DNA and RNA

To determine the effect of single and multiple modifications on duplexstability modified oligodeoxynucleotides ON1-ON11 were prepared,represented in Table 8, and measured T_(m) values of duplexes withcomplementary DNA and RNA by UV-melting curve analysis.

TABLE 8T_(m) and ΔT_(m)/mod data from UV-melting curves (260 nm) of ON1-ON11in duplex with complementary DNA and RNA. T_(m)(° C.) ΔT_(m)/modT_(m)(° C.) ΔT_(m)/mod Entry Sequence^(a,b,c) vs DNA (° C.) vs RNA(° C.) ON1 d(GGATGTTCtCGA) 50.3  1.2 47.6 -1.8 (SEQ ID NO: 1) ON2d(GGAtGTTCTCGA) 49.0 -0.1 47.0 -2.4 (SEQ ID NO: 2) ON3 d(GGATGttCTCGA)46.9 -1.1 45.4 -2.0 (SEQ ID NO: 3) ON4 d(GGATGTTcTCGA) 52.2  3.1 50.4 1.0 (SEQ ID NO: 4) ON5 d(GGATGTTCTcGA) 53.4  4.3 49.5  0.1(SEQ ID NO: 5) ON6 d(GGaTGTTCTCGA) 45.6 -3.5 50.0  0.6 (SEQ ID NO: 6)ON7 d(GGATgTTCTCGA) 46.3 -2.8 50.0  0.6 (SEQ ID NO: 7) ON8d(GGATGTTcTCGA) 50.7  1.6 50.0  0.6 (SEQ ID NO: 8) ON9 d(GGATGTTCTcGA)51.8  2.8 49.2 -0.2 (SEQ ID NO: 9) ON10 d(GGATGTTcTcGA) 51.9  1.4 48.5-0.9 (SEQ ID NO: 10) ON11 d(GCAttt ttACCG)^(d) 33.2 -2.9 27.9 -3.2(SEQ ID NO: 11) ^(a)total strand conc. 2 μM in 10 mM NaH₂PO₄, 150 mMNaCl, pH 7.0. ^(b)A, G, T, C denote natural 2′-deoxynucleosides; a, t,g, c corresponds to modified nucleosides, c stands for the modified5-methyl cytosine nucleoside. ^(c)T_(m) of unmodified duplexes, DNA/DNA:49.1° C., DNA/RNA: 49.4° C. ^(d)T_(m) of unmodified duplexes, DNA/DNA:47.5° C., DNA/RNA: 44.0° C.

According to the ΔT_(m)/mod data, the impact of single or doublemodifications on thermal duplex stability is relatively moderate. Purinemodifications (ON6, 7) tend to destabilize duplexes with complementaryDNA but show a slight increase in T_(m) with complementary RNA. Withinthe pyrimidine series there is, however, more variability. Modifiedthymidine nucleosides (ON1-3) typically lead to a small depression ofT_(m) with RNA as complement while no clear trend with DNA is observed.Interestingly, both cytosine modifications (ON4, 5, 8-10) significantlystabilize duplexes with complementary DNA while having less effect onthe T_(m) with complementary RNA. The stabilizing effect is morepronounced for the 5-methyl cytosine nucleosides, pointing to itspotential to reinforce stacking interactions with neighboringbase-pairs.

To determine the base-pairing selectivity T_(m)s of ON2 in complex withcomplementary DNA having all three possible mismatched bases opposingthe site of modification was measured (Table 9). The T_(m)s of themismatched duplexes are lower by 5.1 to 13° C. with the GT wobble pair,as expected, being least destabilizing. These data compare well with thefully natural mismatched series, indicating no significant change inbase pairing selectivity of the modification.

TABLE 9 T_(m) values from UV-melting curves (260 nm) of ON2 and DNA1in duplex with complementary DNA incorporating one mismatch. EntryDuplex X = A X = T X = G X = C ON2 5′-d(GGA TGT TCt CGA)-3′ 50.3 41.045.2 37.3 (SEQ ID NO: 2) DNA 5′-d(TCG XGA ACA TCC)-3′ (SEQ ID NO: 25)DNA 1 5′-d(GGA TGT TCT CGA)-3′ 49.1 38.3 40.5 36.7 (SEQ ID NO: 26) DNA5′-d(TCG XGA ACA TCC)-3′ (SEQ ID NO: 25) Experimental conditions: totalstrand conc. 2 μM in 10 mM NaH₂PO₄, 150 mM NaCl, pH 7.0

Example 69 Pairing Properties of Fully Modified β-Oligonucleotides withComplementary DNA and RNA

A fully modified nonamer consisting of pyrimidine bases only (ON12) aswell as 3 dodecamers (ON13-15) containing all four bases were preparedto test their affinity to complementary DNA or RNA in either theantiparallel or parallel orientation. Form the corresponding T_(m) data(Table 10) it becomes clear that binding is generally weak. Even underhigh salt (1 M NaCl) conditions, no melting transition could be observedfor ON12 with the antiparallel DNA or RNA complement in the temperaturerange from 5-80° C., suggesting that no duplex was formed. However, withthe longer dodecamers ON13-15, transitions were observed withcomplementary antiparallel DNA and RNA. The T_(m)s of these duplexes areroughly 30° C. lower as compared to the natural control duplexes,amounting to an average ΔT_(m)/mod of −2.5° C. This was not expectedfrom the T_(m) experiments of single incorporations (Table 8) andhighlights the importance of fully modified oligonucleotides whencharacterizing novel pairing systems. Importantly, no signs of duplexformation were found with the corresponding parallel DNA and RNAcomplements. This clearly demonstrates that 7′,5′-bc-DNA is still ableto communicate with the natural nucleic acids via antiparallel duplexstructures, albeit on a substantially lower affinity level.

TABLE 10 T_(m) values from UV-melting curves (260 nm) of fully modifiedON12-ON15 in duplex with complementary antiparallel or parallelDNA or RNA. T_(m)(° C.) T_(m)(° C.) vs DNA vs RNA T_(m)(° C.)T_(m)(° C.) anti- anti- vs DNA vs RNA Entry Sequence parallel parallelparallel parallel ON12 5′-(ttt tct cct)-7′ <10 <10 n.m.^(a) n.m.^(a)ON13 5′-(gga tgt tct cga)-7′  18.9  18.6 <10 <10 ON145′-(tcg aga aca tcc)-7′  15.7 n.d.^(a) <10 <10 ON155′-(cct aca aga gct)-7′  13.1  13.0 <10 n.d.^(a) Experimentalconditions: total strand concentration: 2 μM in 10 mM NaH₂PO₄, 1M NaCl,pH 7.0. ^(a)n.m.: not measured. ^(b)n.d.: not detectable due toformation of a self-structure of the corresponding RNA strand with aT_(m) of 28° C.

Example 70 Self-pairing of 7′,5′-β-bc-DNA

ON14 has been designed to be the antiparallel and ON15 to be theparallel complement to ON13. With this it was possible to investigateself-pairing of 7′,5′-bc-DNA in both orientations. It turned out thatthe antiparallel duplex ON13.ON14 showed a classical sigmoidal meltingbehavior (FIG. 11) with a T_(m) of 54.5° C. This is higher by 5.4° C.compared to the natural duplex of the same sequence. Interestingly thehyperchromicity at 260 nm of the 7′,5′-bc-DNA duplex amounts to only 20%of that of the natural duplex. This indicates significantly differentstacking arrangement of the bases in both duplex conformations. In theparallel orientation (ON13.ON15) no transition could be founddemonstrating the inability of parallel duplex formation in7′,5′-bc-DNA.

Example 71 Thermodynamic Data of Duplex Formation of Beta AnomericOligomers

Transition enthalpies and entropies were determined for the duplexON13.ON14 and the corresponding natural duplex by curve fitting to theexperimental melting curves in analogy to known methods (Table 11)(Petersheim et al., Biochemistry 1983, 22, 256). These data suggest thatthe natural duplex is enthalpically stabilized while the 7′,5′-bc-DNAduplex is entropically favored. This matches with previous findings inthe bc-DNA series and hints to conformational restriction of the7′,5′-bc-DNA backbone as the origin of the entropic stabilization. Thefree enthalpies of duplex formation (ΔG) are in line with the T_(m) datawhich qualify them as a measure for thermodynamic duplex stability.

TABLE 11 Thermodynamic data of duplex formation. 5′-GGA TGT TCT CGA(SEQ ID NO: 26) ΔH ΔS ΔG^(25° C.) CCT ACA AGA GCT-5′ [kcal [cal mo1⁻¹[kcal (SEQ ID NO: 29) mol⁻¹]  K⁻¹] mol⁻¹] DNA•DNA -91.7 -257.9 -14.8(SEQ ID NOS: 26 and 29) ON13•ON14 -77.0 -208.0 -15.0 (SEQ ID NOS: 13 and14)

Example 72 CD-Spectroscopy of Beta Anomers of Beta Anomeric Oligomers

To gain insight into the structural properties of the homo 7′,5′-bc-DNAduplex and the hybrid 7′,5′-bc-DNA/DNA duplex CD-spectra were measuredand compared with that of the corresponding natural DNA duplex (FIG.12). As expected, the natural duplex shows the classical features of aB-DNA duplex. The homo 7′,5′-bc-DNA duplex, however, has a CD signaturethat is significantly different from that of A- or B-type doublehelices. It is characterized by two minimum ellipticities around 215 and265 nm with a maximum at 243 nm. The hybrid duplex carries a signaturewhich is closer to that of the DNA duplex. Compared to B-DNA, theminimum ellipticity at 260 nm is red shifted by about 15 nm while themaximum ellipticity at 288 nm is red-shifted by about 7 nm andassociated with a loss in amplitude. The differences in the three CDspectra thus indicate differential arrangements of the bases within thebase-stack, induced by the change in backbone geometry of each strand.This is in agreement with the differential hyperchromicities of the twopairing systems in the corresponding UV-melting curves (FIG. 11).

Example 73 X-Ray Crystal Structure of a 7′,5′-α-Bc-DNA Monomer

A 7′,5′-bc-T analogue was prepared, carrying a p-nitrobenzoate group atO7′. This compound could be crystallized and its structure solved (FIG.10). Besides establishing final proof for the relative configuration ofthe 7′,5′-bc-DNA series we also obtained information on the preferredconformation of the bicyclic sugar scaffold. It clearly emerges that thefuranose part exists in a perfect 1′-exo conformation while thecarbocyclic ring occurs in the 6′-endo configuration, thus placing theoxy-substituent at C7′ in a pseudoaxial and the 5′OH group in apseudoequatorial position. This structure matches exactly one of the twolow energy conformers determined by ab initio calculations.

Compound 33:

A colorless transparent crystal of [C₁₉H₁₉N₃O₈] was mounted in air andused for X-ray structure determination at ambient conditions. Allmeasurements were made on an Oxford Diffraction SuperNova area-detectordiffractometer (Oxford Diffraction (2010)) using mirror opticsmonochromated Mo Kα radiation (λ=0.71073 Å) and Al filtered (Macchi etal., J. Appl. Cryst 2011, 44, 763.). The unit cell constants and anorientation matrix for data collection were obtained from aleast-squares refinement of the setting angles of reflections in therange 2.°<θ<27.°. A total of 530 frames were collected using w scans,with 2.5+2.5 seconds exposure time, a rotation angle of 1° per frame, acrystal-detector distance of 65.1 mm, at T=173(2) K. Data reduction wasperformed using the CrysAlisPro (Version 1.171.34.44). OxfordDiffraction Ltd., Y., Oxfordshire, UK) program. The intensities werecorrected for Lorentz and polarization effects, and an absorptioncorrection based on the multi-scan method using SCALES ABSPACK inCrysAlisPro was applied. Data collection and refinement parameters aregiven in Table 12. The structure was solved by direct methods usingSHELXS-97 (Sheldrick, Acta Cryst. 2008, A64, 112-122), which revealedthe positions of all non-hydrogen atoms of the title compound. Thenon-hydrogen atoms were refined anisotropically. All H-atoms were placedin geometrically calculated positions and refined using a riding modelwhere each H-atom was assigned a fixed isotropic displacement parameterwith a value equal to 1.2 Ueq of its parent atom. Refinement of thestructure was carried out on F² using full-matrix least-squaresprocedures, which minimized the function Σw(F_(o) ²−F_(c) ²)². Theweighting scheme was based on counting statistics and included a factorto down weight the intense reflections. All calculations were performedusing the SHELXL-97 program (Macchi et al., J. Appl. Cryst 2011, 44,763).

TABLE 12 Crystal data and structure refinement for compound 33.Identification code shelx Empirical formula C19H19N3O8 Formula weight417.37 Temperature 173(2) K Wavelength 0.71073 Å Crystal systemMonoclinic Space group P 21 Unit cell dimensions a = 7.0712(2) Å α = 90°b = 6.48123(16) Å β = 98.062(3)° c = 20.4085(6) Å γ = 90° Volume926.08(4) Å3 Z 2 Density (calculated) 1.497 Mg/m³ Absorption coefficient0.119 mm⁻¹ F(000) 436 Crystal size 0.5326 × 0.308 × 0.1151 mm³ Thetarange for data 2.016 to 27.110° collection Index ranges −8 <= h <= 8, −7<= k <= 8, −24 <= l <= 24 Reflections collected 6928 Independentreflections 3651 [R(int) = 0.0200] Completeness to 99.8% theta = 25.000°Absorption correction Gaussian Max. and min. 0.987 and 0.963transmission Refinement method Full-matrix least- squares on F²Data/restraints/ 3651/1/274 parameters Goodness-of-fit on F² 1.059 FinalR indices R1 = 0.0344, wR2 = 0.0772 [I > 2sigma(I)] R indices (all data)R1 = 0.0387, wR2 = 0.0808 Absolute structure −0.9(5) parameterExtinction coefficient 0.009(2) Largest diff. peak 0.212 and −0.236e.Å⁻³ and hole

What is claimed is:
 1. A compound of formula (I):

wherein T₁ and T₂ are each independently selected from the groupconsisting of OR₁ and OR₂; and; wherein R₁ is H or a hydroxyl protectinggroup, and R₂ is a phosphorus moiety; and wherein Bx is a nucleobase. 2.The compound of claim 1, having the structure of formula (II)

wherein T₁ and T₂ are each independently selected from the groupconsisting of OR₁ and OR₂.
 3. The compound of claim 1, having thestructure of formula (III)

wherein T₁ and T₂ are each independently selected from the groupconsisting of OR₁ and OR₂.
 4. The compound of claim 1, wherein saidphosphorus moiety R₂ is selected from the group consisting of aphosphate moiety, a phosphoramidate moiety and a phosphoramidite moiety.5. The compound of claim 1, having the structure selected from the groupconsisting of:


6. The compound of claim 1, wherein the nucleobase is selected from thegroup consisting of a purine base and a pyrimidine base.
 7. An oligomercomprising at least one monomer subunit as a compound of formula (IV)

wherein either T₃ or T₄ are independently a nucleosidic linkage group;and when T₃ is a nucleosidic linkage group; T₄ is selected from thegroup consisting of OR₁, OR₂, a 5′ terminal group, a 7′ terminal groupand a nucleosidic linkage group, and when T₄ is a nucleosidic linkagegroup; T₃ is selected from the group consisting of OR₁, OR₂, a 5′terminal group, a 7′ terminal group and a nucleosidic linkage group,and; wherein R₁ is selected from H and a hydroxyl protecting group, andR₂ is a phosphorus moiety; and Bx is a nucleobase.
 8. The oligomer ofclaim 7, having the structure of formula (V):

wherein: (i) when T₃ is a nucleosidic linkage group, T₄ is selected fromthe group consisting of a 7′ terminal group, OR₁,and OR₂; or (ii) whenT₃ is selected from the group consisting of a 5′ terminal group, OR₁,and OR₂, T₄ is a nucleosidic linkage group; or (iii) T₃ and T₄ areindependently of each other a nucleosidic linkage group.
 9. The oligomerof claim 7, having the structure of formula (VI):

wherein: (i) when T₃ is a nucleosidic linkage group, T₄ is selected fromthe group consisting of a 7′ terminal group, OR₁,and OR₂; or (ii) whenT₃ is selected from the group consisting of a 5′ terminal group, OR₁,and OR₂, T₄ is a nucleosidic linkage group; or (iii) T₃ and T₄ areindependently of each other a nucleosidic linkage group.
 10. Theoligomer of claim 7, wherein each nucleosidic linkage group isindependently selected from the group consisting of a phosphodiesterlinkage group, a phosphotriester linkage group, a phosphorothioatelinkage group, a phosphorodithioate linkage group, a phosphonate linkagegroup, a phosphonothioate linkage group, a phosphinate linkage group, aphosphorthioamidate linkage and a phosphoramidate linkage group.
 11. Theoligomer of claim 10 wherein each nucleosidic linkage group isindependently selected from the group consisting of a phosphodiesterlinkage group and a phosphorothioate linkage group.
 12. The oligomer ofclaim 11 wherein each nucleosidic linkage group is a phosphorothioatelinkage group.
 13. The oligomer of claim 11, wherein each nucleosidiclinkage group is a phosphodiester linkage group.
 14. The oligomer ofclaim 7, comprising at least two contiguous monomer subunits ofcompounds of formula (IV), wherein each of the contiguous subunit ofcompound of formula (IV) is independently linked to the adjacentcontiguous subunit of compound of formula (IV) by the nucleosidiclinkage group, wherein the nucleosidic linkage group links a 5′ terminusand a 7′ terminus of the two contiguous subunits of compounds of formula(IV).
 15. The oligomer of claim 7, wherein said oligomer comprises atleast one nucleic acid sequence, wherein said nucleic acid sequencecomprises said at least one monomer subunit of compound of formula (IV),and wherein said nucleic acid sequence is selected from the groupconsisting of the SEQ ID NO: 1 to
 24. 16. The oligomer of claim 15,wherein the nucleic acid sequence is SEQ ID NO:
 24. 17. The oligomer ofclaim 7, wherein the oligomer comprises a nucleic acid sequence, whereinsaid nucleic acid sequence consists of at least two contiguous monomersubunits of compounds of formula (V), wherein said nucleic acid sequenceis flanked on its 5′ terminus and on its 7′ terminus by at least onenucleotide or nucleoside that is different from the compound of formula(IV), wherein the 5′ terminus of said nucleic acid sequence is linked toa 5′ terminus of the nucleotide or nucleoside that is different from thecompound of formula (IV), and wherein the 7′ terminus of said nucleicacid sequence is linked to a 3′ terminus of the nucleotide or nucleosidethat is different from the compound of formula (IV).
 18. The oligomer ofclaim 7, wherein the oligomer comprises a nucleic acid sequence, whereinsaid nucleic acid sequence consists of at least two contiguous monomersubunits of compounds of formula (VI), wherein said nucleic acidsequence is flanked on its 5′ terminus and on its 7′ terminus each by atleast one nucleotide or nucleoside that is different from the compoundof formula (IV), wherein the 5′ terminus of said nucleic acid sequenceis linked to a 3′ terminus of the nucleotide or nucleoside that isdifferent from the compound of formula (IV), and wherein the 7′ terminusof said nucleic acid sequence is linked to a 5′ terminus of thenucleotide or nucleoside that is different from the compound of formula(IV).
 19. The oligomer of claim 7, wherein said monomer subunit ofcompound of formula (IV) is selected from the group consisting of:


20. The oligomer of claim 7, wherein the nucleobase is selected from thegroup consisting of a purine base and a pyrimidine base.